Methods and compositions for diagnosis and prognosis of renal injury and renal failure

ABSTRACT

The present invention relates to methods and compositions for monitoring, diagnosis, prognosis, and determination of treatment regimens in subjects suffering from or suspected of having a renal injury. In particular, the invention relates to using a plurality of assays configured to detect a kidney injury marker as diagnostic and prognostic biomarkers in renal injuries.

This application is a divisional application of U.S. patent applicationSer. No. 13/580,878, filed Oct. 23, 2012, now U.S. Pat. No. 9,029,093,issued May 12, 2015, which is the U.S. national phase of InternationalApplication No. PCT/US2011/026384, filed Sep. 26, 2011, which designatedthe U.S. and claims the benefit of priority to U.S. Provisional PatentApplication No. 61/308,861 filed Feb. 26, 2010, each of which is herebyincorporated in its entirety including all tables, figures, and claims.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

The kidney is responsible for water and solute excretion from the body.Its functions include maintenance of acid-base balance, regulation ofelectrolyte concentrations, control of blood volume, and regulation ofblood pressure. As such, loss of kidney function through injury and/ordisease results in substantial morbidity and mortality. A detaileddiscussion of renal injuries is provided in Harrison's Principles ofInternal Medicine, 17^(th) Ed., McGraw Hill, N.Y., pages 1741-1830,which are hereby incorporated by reference in their entirety. Renaldisease and/or injury may be acute or chronic. Acute and chronic kidneydisease are described as follows (from Current Medical Diagnosis &Treatment 2008, 47^(th) Ed, McGraw Hill, N.Y., pages 785-815, which arehereby incorporated by reference in their entirety): “Acute renalfailure is worsening of renal function over hours to days, resulting inthe retention of nitrogenous wastes (such as urea nitrogen) andcreatinine in the blood. Retention of these substances is calledazotemia. Chronic renal failure (chronic kidney disease) results from anabnormal loss of renal function over months to years”.

Acute renal failure (ARF, also known as acute kidney injury, or AKI) isan abrupt (typically detected within about 48 hours to 1 week) reductionin glomerular filtration. This loss of filtration capacity results inretention of nitrogenous (urea and creatinine) and non-nitrogenous wasteproducts that are normally excreted by the kidney, a reduction in urineoutput, or both. It is reported that ARF complicates about 5% ofhospital admissions, 4-15% of cardiopulmonary bypass surgeries, and upto 30% of intensive care admissions. ARF may be categorized as prerenal,intrinsic renal, or postrenal in causation. Intrinsic renal disease canbe further divided into glomerular, tubular, interstitial, and vascularabnormalities. Major causes of ARF are described in the following table,which is adapted from the Merck Manual, 17^(th) ed., Chapter 222, andwhich is hereby incorporated by reference in their entirety.

TABLE 1 Type Risk Factors Prerenal ECF volume depletion Excessivediuresis, hemorrhage, GI losses, loss of intravascular fluid into theextravascular space (due to ascites, peritonitis, pancreatitis, orburns), loss of skin and mucus membranes, renal salt- and water-wastingstates Low cardiac output Cardiomyopathy, MI, cardiac tamponade,pulmonary embolism, pulmonary hypertension, positive-pressure mechanicalventilation Low systemic vascular Septic shock, liver failure,antihypertensive drugs resistance Increased renal vascular NSAIDs,cyclosporines, tacrolimus, hypercalcemia, resistance anaphylaxis,anesthetics, renal artery obstruction, renal vein thrombosis, sepsis,hepatorenal syndrome Decreased efferent ACE inhibitors or angiotensin IIreceptor blockers arteriolar tone (leading to decreased GFR from reducedglomerular transcapillary pressure, especially in patients withbilateral renal artery stenosis) Intrinsic Renal Acute tubular injuryIschemia (prolonged or severe prerenal state): surgery, hemorrhage,arterial or venous obstruction; Toxins: NSAIDs, cyclosporines,tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin,myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrastagents, streptozotocin Acute glomerulonephritis ANCA-associated:Crescentic glomerulonephritis, polyarteritis nodosa, Wegener'sgranulomatosis; Anti-GBM glomerulonephritis: Goodpasture's syndrome;Immune-complex: Lupus glomerulonephritis, postinfectiousglomerulonephritis, cryoglobulinemic glomerulonephritis Acutetubulointerstitial Drug reaction (eg, β-lactams, NSAIDs, nephritissulfonamides, ciprofloxacin, thiazide diuretics, , furosemide phenytoin,allopurinol, pyelonephritis, papillary necrosis Acute vascularVasculitis, malignant hypertension, thrombotic nephropathymicroangiopathies, scleroderma, atheroembolism Infiltrative diseasesLymphoma, sarcoidosis, leukemia Postrenal Tubular precipitation Uricacid (tumor lysis), sulfonamides, triamterene, acyclovir, indinavir,methotrexate, ethylene glycol ingestion, myeloma protein, myoglobinUreteral obstruction Intrinsic: Calculi, clots, sloughed renal tissue,fungus ball, edema, malignancy, congenital defects; Extrinsic:Malignancy, retroperitoneal fibrosis, ureteral trauma during surgery orhigh impact injury Bladder obstruction Mechanical: Benign prostatichyperplasia, prostate cancer, bladder cancer, urethral strictures,phimosis, paraphimosis, urethral valves, obstructed indwelling urinarycatheter; Neurogenic: Anticholinergic drugs, upper or lower motor neuronlesion

In the case of ischemic ARF, the course of the disease may be dividedinto four phases. During an initiation phase, which lasts hours to days,reduced perfusion of the kidney is evolving into injury. Glomerularultrafiltration reduces, the flow of filtrate is reduced due to debriswithin the tubules, and back leakage of filtrate through injuredepithelium occurs. Renal injury can be mediated during this phase byreperfusion of the kidney. Initiation is followed by an extension phasewhich is characterized by continued ischemic injury and inflammation andmay involve endothelial damage and vascular congestion. During themaintenance phase, lasting from 1 to 2 weeks, renal cell injury occurs,and glomerular filtration and urine output reaches a minimum. A recoveryphase can follow in which the renal epithelium is repaired and GFRgradually recovers. Despite this, the survival rate of subjects with ARFmay be as low as about 60%.

Acute kidney injury caused by radiocontrast agents (also called contrastmedia) and other nephrotoxins such as cyclosporine, antibioticsincluding aminoglycosides and anticancer drugs such as cisplatinmanifests over a period of days to about a week. Contrast inducednephropathy (CIN, which is AKI caused by radiocontrast agents) isthought to be caused by intrarenal vasoconstriction (leading to ischemicinjury) and from the generation of reactive oxygen species that aredirectly toxic to renal tubular epithelial cells. CIN classicallypresents as an acute (onset within 24-48 h) but reversible (peak 3-5days, resolution within 1 week) rise in blood urea nitrogen and serumcreatinine.

A commonly reported criteria for defining and detecting AKI is an abrupt(typically within about 2-7 days or within a period of hospitalization)elevation of serum creatinine. Although the use of serum creatinineelevation to define and detect AKI is well established, the magnitude ofthe serum creatinine elevation and the time over which it is measured todefine AKI varies considerably among publications. Traditionally,relatively large increases in serum creatinine such as 100%, 200%, anincrease of at least 100% to a value over 2 mg/dL and other definitionswere used to define AKI. However, the recent trend has been towardsusing smaller serum creatinine rises to define AKI. The relationshipbetween serum creatinine rise, AKI and the associated health risks arereviewed in Praught and Shlipak, Curr Opin Nephrol Hypertens 14:265-270,2005 and Chertow et al, J Am Soc Nephrol 16: 3365-3370, 2005, which,with the references listed therein, are hereby incorporated by referencein their entirety. As described in these publications, acute worseningrenal function (AKI) and increased risk of death and other detrimentaloutcomes are now known to be associated with very small increases inserum creatinine. These increases may be determined as a relative(percent) value or a nominal value. Relative increases in serumcreatinine as small as 20% from the pre-injury value have been reportedto indicate acutely worsening renal function (AKI) and increased healthrisk, but the more commonly reported value to define AKI and increasedhealth risk is a relative increase of at least 25%. Nominal increases assmall as 0.3 mg/dL, 0.2 mg/dL or even 0.1 mg/dL have been reported toindicate worsening renal function and increased risk of death. Varioustime periods for the serum creatinine to rise to these threshold valueshave been used to define AKI, for example, ranging from 2 days, 3 days,7 days, or a variable period defined as the time the patient is in thehospital or intensive care unit. These studies indicate there is not aparticular threshold serum creatinine rise (or time period for the rise)for worsening renal function or AKI, but rather a continuous increase inrisk with increasing magnitude of serum creatinine rise.

One study (Lassnigg et all, J Am Soc Nephrol 15:1597-1605, 2004, herebyincorporated by reference in its entirety) investigated both increasesand decreases in serum creatinine. Patients with a mild fall in serumcreatinine of −0.1 to −0.3 mg/dL following heart surgery had the lowestmortality rate. Patients with a larger fall in serum creatinine (morethan or equal to −0.4 mg/dL) or any increase in serum creatinine had alarger mortality rate. These findings caused the authors to concludethat even very subtle changes in renal function (as detected by smallcreatinine changes within 48 hours of surgery) seriously effectpatient's outcomes. In an effort to reach consensus on a unifiedclassification system for using serum creatinine to define AKI inclinical trials and in clinical practice, Bellomo et al., Crit Care.8(4):R204-12, 2004, which is hereby incorporated by reference in itsentirety, proposes the following classifications for stratifying AKIpatients:

-   “Risk”: serum creatinine increased 1.5 fold from baseline OR urine    production of <0.5 ml/kg body weight/hr for 6 hours;-   “Injury”: serum creatinine increased 2.0 fold from baseline OR urine    production <0.5 ml/kg/hr for 12 h;-   “Failure”: serum creatinine increased 3.0 fold from baseline OR    creatinine >355 μmol/l (with a rise of >44) or urine output below    0.3 ml/kg/hr for 24 h or anuria for at least 12 hours;-   And included two clinical outcomes:-   “Loss”: persistent need for renal replacement therapy for more than    four weeks.-   “ESRD”: end stage renal disease—the need for dialysis for more than    3 months.-   These criteria are called the RIFLE criteria, which provide a useful    clinical tool to classify renal status. As discussed in Kellum,    Crit. Care Med. 36: S141-45, 2008 and Ricci et al., Kidney Int. 73,    538-546, 2008, each hereby incorporated by reference in its    entirety, the RIFLE criteria provide a uniform definition of AKI    which has been validated in numerous studies.

More recently, Mehta et al., Crit. Care 11:R31 (doi:10.1186.cc5713),2007, hereby incorporated by reference in its entirety, proposes thefollowing similar classifications for stratifying AKI patients, whichhave been modified from RIFLE:

-   “Stage I”: increase in serum creatinine of more than or equal to 0.3    mg/dL (≧26.4 μmol/L) or increase to more than or equal to 150%    (1.5-fold) from baseline OR urine output less than 0.5 mL/kg per    hour for more than 6 hours;-   “Stage II”: increase in serum creatinine to more than 200% (>2-fold)    from baseline OR urine output less than 0.5 mL/kg per hour for more    than 12 hours;-   “Stage III”: increase in serum creatinine to more than 300%    (>3-fold) from baseline OR serum creatinine ≧354 μmol/L accompanied    by an acute increase of at least 44 μmol/L OR urine output less than    0.3 mL/kg per hour for 24 hours or anuria for 12 hours.

The CIN Consensus Working Panel (McCollough et al, Rev Cardiovasc Med.2006; 7(4):177-197, hereby incorporated by reference in its entirety)uses a serum creatinine rise of 25% to define Contrast inducednephropathy (which is a type of AKI).Although various groups proposeslightly different criteria for using serum creatinine to detect AKI,the consensus is that small changes in serum creatinine, such as 0.3mg/dL or 25%, are sufficient to detect AKI (worsening renal function)and that the magnitude of the serum creatinine change is an indicator ofthe severity of the AKI and mortality risk.

Although serial measurement of serum creatinine over a period of days isan accepted method of detecting and diagnosing AKI and is considered oneof the most important tools to evaluate AKI patients, serum creatinineis generally regarded to have several limitations in the diagnosis,assessment and monitoring of AKI patients. The time period for serumcreatinine to rise to values (e.g., a 0.3 mg/dL or 25% rise) considereddiagnostic for AKI can be 48 hours or longer depending on the definitionused. Since cellular injury in AKI can occur over a period of hours,serum creatinine elevations detected at 48 hours or longer can be a lateindicator of injury, and relying on serum creatinine can thus delaydiagnosis of AKI. Furthermore, serum creatinine is not a good indicatorof the exact kidney status and treatment needs during the most acutephases of AKI when kidney function is changing rapidly. Some patientswith AKI will recover fully, some will need dialysis (either short termor long term) and some will have other detrimental outcomes includingdeath, major adverse cardiac events and chronic kidney disease. Becauseserum creatinine is a marker of filtration rate, it does notdifferentiate between the causes of AKI (pre-renal, intrinsic renal,post-renal obstruction, atheroembolic, etc) or the category or locationof injury in intrinsic renal disease (for example, tubular, glomerularor interstitial in origin). Urine output is similarly limited, Knowingthese things can be of vital importance in managing and treatingpatients with AKI.

These limitations underscore the need for better methods to detect andassess AKI, particularly in the early and subclinical stages, but alsoin later stages when recovery and repair of the kidney can occur.Furthermore, there is a need to better identify patients who are at riskof having an AKI.

BRIEF SUMMARY OF THE INVENTION

It is an object of the invention to provide methods and compositions forevaluating renal function in a subject. As described herein, measurementof a plurality of assays, wherein one or more of the assays isconfigured to detect one or more of the biomarkers listed in Table 2herein (collectively referred to herein as “kidney injury markers, andeach individually as a “kidney injury marker”) The plurality of assaysare combined to provide a “biomarker panel approach” which can be usedfor diagnosis, prognosis, risk stratification, staging, monitoring,categorizing and determination of further diagnosis and treatmentregimens in subjects suffering or at risk of suffering from an injury torenal function, reduced renal function, and/or acute renal failure (alsocalled acute kidney injury). Preferred biomarker panels comprise two,three, four, five, or more assays configured to detect two, three, four,five, or more of the biomarkers listed in Table 2 herein.

These kidney injury markers may be used in panels comprising a pluralityof kidney injury markers, for risk stratification (that is, to identifysubjects at risk for a future injury to renal function, for futureprogression to reduced renal function, for future progression to ARF,for future improvement in renal function, etc.); for diagnosis ofexisting disease (that is, to identify subjects who have suffered aninjury to renal function, who have progressed to reduced renal function,who have progressed to ARF, etc.); for monitoring for deterioration orimprovement of renal function; and for predicting a future medicaloutcome, such as improved or worsening renal function, a decreased orincreased mortality risk, a decreased or increased risk that a subjectwill require renal replacement therapy (i.e., hemodialysis, peritonealdialysis, hemofiltration, and/or renal transplantation, a decreased orincreased risk that a subject will recover from an injury to renalfunction, a decreased or increased risk that a subject will recover fromARF, a decreased or increased risk that a subject will progress to endstage renal disease, a decreased or increased risk that a subject willprogress to chronic renal failure, a decreased or increased risk that asubject will suffer rejection of a transplanted kidney, etc.

In a first aspect, the present invention relates to methods forevaluating renal status in a subject. These methods comprise performingan assay method that is configured to detect one or more kidney injurymarkers of the present invention in a body fluid sample obtained fromthe subject. A plurality of assay results, for example comprising ameasured concentration of one or more markers selected from the groupconsisting of the markers listed in Table 2 herein are then correlatedto the renal status of the subject. This correlation to renal status mayinclude correlating the assay result(s) to one or more of riskstratification, diagnosis, prognosis, staging, classifying andmonitoring of the subject as described herein. Thus, the presentinvention utilizes one or more kidney injury markers of the presentinvention for the evaluation of renal injury.

In certain embodiments, the methods for evaluating renal statusdescribed herein are methods for risk stratification of the subject;that is, assigning a likelihood of one or more future changes in renalstatus to the subject. In these embodiments, the assay result(s) is/arecorrelated to one or more such future changes. The following arepreferred risk stratification embodiments.

In preferred risk stratification embodiments, these methods comprisedetermining a subject's risk for a future injury to renal function, andthe assay result(s) is/are correlated to a likelihood of such a futureinjury to renal function. For example, the measured concentration(s) mayeach be compared to a threshold value. For a “positive going” kidneyinjury marker, an increased likelihood of suffering a future injury torenal function is assigned to the subject when the measuredconcentration is above the threshold, relative to a likelihood assignedwhen the measured concentration is below the threshold. For a “negativegoing” kidney injury marker, an increased likelihood of suffering afuture injury to renal function is assigned to the subject when themeasured concentration is below the threshold, relative to a likelihoodassigned when the measured concentration is above the threshold.

In other preferred risk stratification embodiments, these methodscomprise determining a subject's risk for future reduced renal function,and the assay result(s) is/are correlated to a likelihood of suchreduced renal function. For example, the measured concentrations mayeach be compared to a threshold value. For a “positive going” kidneyinjury marker, an increased likelihood of suffering a future reducedrenal function is assigned to the subject when the measuredconcentration is above the threshold, relative to a likelihood assignedwhen the measured concentration is below the threshold. For a “negativegoing” kidney injury marker, an increased likelihood of future reducedrenal function is assigned to the subject when the measuredconcentration is below the threshold, relative to a likelihood assignedwhen the measured concentration is above the threshold.

In still other preferred risk stratification embodiments, these methodscomprise determining a subject's likelihood for a future improvement inrenal function, and the assay result(s) is/are correlated to alikelihood of such a future improvement in renal function. For example,the measured concentration(s) may each be compared to a threshold value.For a “positive going” kidney injury marker, an increased likelihood ofa future improvement in renal function is assigned to the subject whenthe measured concentration is below the threshold, relative to alikelihood assigned when the measured concentration is above thethreshold. For a “negative going” kidney injury marker, an increasedlikelihood of a future improvement in renal function is assigned to thesubject when the measured concentration is above the threshold, relativeto a likelihood assigned when the measured concentration is below thethreshold.

In yet other preferred risk stratification embodiments, these methodscomprise determining a subject's risk for progression to ARF, and theresult(s) is/are correlated to a likelihood of such progression to ARF.For example, the measured concentration(s) may each be compared to athreshold value. For a “positive going” kidney injury marker, anincreased likelihood of progression to ARF is assigned to the subjectwhen the measured concentration is above the threshold, relative to alikelihood assigned when the measured concentration is below thethreshold. For a “negative going” kidney injury marker, an increasedlikelihood of progression to ARF is assigned to the subject when themeasured concentration is below the threshold, relative to a likelihoodassigned when the measured concentration is above the threshold.

And in other preferred risk stratification embodiments, these methodscomprise determining a subject's outcome risk, and the assay result(s)is/are correlated to a likelihood of the occurrence of a clinicaloutcome related to a renal injury suffered by the subject. For example,the measured concentration(s) may each be compared to a threshold value.For a “positive going” kidney injury marker, an increased likelihood ofone or more of: acute kidney injury, progression to a worsening stage ofAKI, mortality, a requirement for renal replacement therapy, arequirement for withdrawal of renal toxins, end stage renal disease,heart failure, stroke, myocardial infarction, progression to chronickidney disease, etc., is assigned to the subject when the measuredconcentration is above the threshold, relative to a likelihood assignedwhen the measured concentration is below the threshold. For a “negativegoing” kidney injury marker, an increased likelihood of one or more of:acute kidney injury, progression to a worsening stage of AKI, mortality,a requirement for renal replacement therapy, a requirement forwithdrawal of renal toxins, end stage renal disease, heart failure,stroke, myocardial infarction, progression to chronic kidney disease,etc., is assigned to the subject when the measured concentration isbelow the threshold, relative to a likelihood assigned when the measuredconcentration is above the threshold.

In such risk stratification embodiments, preferably the likelihood orrisk assigned is that an event of interest is more or less likely tooccur within 180 days of the time at which the body fluid sample isobtained from the subject. In particularly preferred embodiments, thelikelihood or risk assigned relates to an event of interest occurringwithin a shorter time period such as 18 months, 120 days, 90 days, 60days, 45 days, 30 days, 21 days, 14 days, 7 days, 5 days, 96 hours, 72hours, 48 hours, 36 hours, 24 hours, 12 hours, or less. A risk at 0hours of the time at which the body fluid sample is obtained from thesubject is equivalent to diagnosis of a current condition.

In preferred risk stratification embodiments, the subject is selectedfor risk stratification based on the pre-existence in the subject of oneor more known risk factors for prerenal, intrinsic renal, or postrenalARF. For example, a subject undergoing or having undergone majorvascular surgery, coronary artery bypass, or other cardiac surgery; asubject having pre-existing congestive heart failure, preeclampsia,eclampsia, diabetes mellitus, hypertension, coronary artery disease,proteinuria, renal insufficiency, glomerular filtration below the normalrange, cirrhosis, serum creatinine above the normal range, or sepsis; ora subject exposed to NSAIDs, cyclosporines, tacrolimus, aminoglycosides,foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavymetals, methotrexate, radiopaque contrast agents, or streptozotocin areall preferred subjects for monitoring risks according to the methodsdescribed herein. This list is not meant to be limiting. By“pre-existence” in this context is meant that the risk factor exists atthe time the body fluid sample is obtained from the subject. Inparticularly preferred embodiments, a subject is chosen for riskstratification based on an existing diagnosis of injury to renalfunction, reduced renal function, or ARF.

In other embodiments, the methods for evaluating renal status describedherein are methods for diagnosing a renal injury in the subject; thatis, assessing whether or not a subject has suffered from an injury torenal function, reduced renal function, or ARF. In these embodiments,the assay results, for example comprising a measured concentration ofone or more markers selected from the group consisting of the biomarkerslisted in Table 2 herein are correlated to the occurrence ornonoccurrence of a change in renal status. The following are preferreddiagnostic embodiments.

In preferred diagnostic embodiments, these methods comprise diagnosingthe occurrence or nonoccurrence of an injury to renal function, and theassay result(s) is/are correlated to the occurrence or nonoccurrence ofsuch an injury. For example, each of the measured concentration(s) maybe compared to a threshold value. For a positive going marker, anincreased likelihood of the occurrence of an injury to renal function isassigned to the subject when the measured concentration is above thethreshold (relative to the likelihood assigned when the measuredconcentration is below the threshold); alternatively, when the measuredconcentration is below the threshold, an increased likelihood of thenonoccurrence of an injury to renal function may be assigned to thesubject (relative to the likelihood assigned when the measuredconcentration is above the threshold). For a negative going marker, anincreased likelihood of the occurrence of an injury to renal function isassigned to the subject when the measured concentration is below thethreshold (relative to the likelihood assigned when the measuredconcentration is above the threshold); alternatively, when the measuredconcentration is above the threshold, an increased likelihood of thenonoccurrence of an injury to renal function may be assigned to thesubject (relative to the likelihood assigned when the measuredconcentration is below the threshold).

In other preferred diagnostic embodiments, these methods comprisediagnosing the occurrence or nonoccurrence of reduced renal function,and the assay result(s) is/are correlated to the occurrence ornonoccurrence of an injury causing reduced renal function. For example,each of the measured concentration(s) may be compared to a thresholdvalue. For a positive going marker, an increased likelihood of theoccurrence of an injury causing reduced renal function is assigned tothe subject when the measured concentration is above the threshold(relative to the likelihood assigned when the measured concentration isbelow the threshold); alternatively, when the measured concentration isbelow the threshold, an increased likelihood of the nonoccurrence of aninjury causing reduced renal function may be assigned to the subject(relative to the likelihood assigned when the measured concentration isabove the threshold). For a negative going marker, an increasedlikelihood of the occurrence of an injury causing reduced renal functionis assigned to the subject when the measured concentration is below thethreshold (relative to the likelihood assigned when the measuredconcentration is above the threshold); alternatively, when the measuredconcentration is above the threshold, an increased likelihood of thenonoccurrence of an injury causing reduced renal function may beassigned to the subject (relative to the likelihood assigned when themeasured concentration is below the threshold).

In yet other preferred diagnostic embodiments, these methods comprisediagnosing the occurrence or nonoccurrence of ARF, and the assayresult(s) is/are correlated to the occurrence or nonoccurrence of aninjury causing ARF. For example, each of the measured concentration(s)may be compared to a threshold value. For a positive going marker, anincreased likelihood of the occurrence of ARF is assigned to the subjectwhen the measured concentration is above the threshold (relative to thelikelihood assigned when the measured concentration is below thethreshold); alternatively, when the measured concentration is below thethreshold, an increased likelihood of the nonoccurrence of ARF may beassigned to the subject (relative to the likelihood assigned when themeasured concentration is above the threshold). For a negative goingmarker, an increased likelihood of the occurrence of ARF is assigned tothe subject when the measured concentration is below the threshold(relative to the likelihood assigned when the measured concentration isabove the threshold); alternatively, when the measured concentration isabove the threshold, an increased likelihood of the nonoccurrence of ARFmay be assigned to the subject (relative to the likelihood assigned whenthe measured concentration is below the threshold).

In still other preferred diagnostic embodiments, these methods comprisediagnosing a subject as being in need of renal replacement therapy, andthe assay result(s) is/are correlated to a need for renal replacementtherapy. For example, each of the measured concentration(s) may becompared to a threshold value. For a positive going marker, an increasedlikelihood of the occurrence of an injury creating a need for renalreplacement therapy is assigned to the subject when the measuredconcentration is above the threshold (relative to the likelihoodassigned when the measured concentration is below the threshold);alternatively, when the measured concentration is below the threshold,an increased likelihood of the nonoccurrence of an injury creating aneed for renal replacement therapy may be assigned to the subject(relative to the likelihood assigned when the measured concentration isabove the threshold). For a negative going marker, an increasedlikelihood of the occurrence of an injury creating a need for renalreplacement therapy is assigned to the subject when the measuredconcentration is below the threshold (relative to the likelihoodassigned when the measured concentration is above the threshold);alternatively, when the measured concentration is above the threshold,an increased likelihood of the nonoccurrence of an injury creating aneed for renal replacement therapy may be assigned to the subject(relative to the likelihood assigned when the measured concentration isbelow the threshold).

In still other preferred diagnostic embodiments, these methods comprisediagnosing a subject as being in need of renal transplantation, and theassay result(s0 is/are correlated to a need for renal transplantation.For example, each of the measured concentration(s) may be compared to athreshold value. For a positive going marker, an increased likelihood ofthe occurrence of an injury creating a need for renal transplantation isassigned to the subject when the measured concentration is above thethreshold (relative to the likelihood assigned when the measuredconcentration is below the threshold); alternatively, when the measuredconcentration is below the threshold, an increased likelihood of thenonoccurrence of an injury creating a need for renal transplantation maybe assigned to the subject (relative to the likelihood assigned when themeasured concentration is above the threshold). For a negative goingmarker, an increased likelihood of the occurrence of an injury creatinga need for renal transplantation is assigned to the subject when themeasured concentration is below the threshold (relative to thelikelihood assigned when the measured concentration is above thethreshold); alternatively, when the measured concentration is above thethreshold, an increased likelihood of the nonoccurrence of an injurycreating a need for renal transplantation may be assigned to the subject(relative to the likelihood assigned when the measured concentration isbelow the threshold).

In still other embodiments, the methods for evaluating renal statusdescribed herein are methods for monitoring a renal injury in thesubject; that is, assessing whether or not renal function is improvingor worsening in a subject who has suffered from an injury to renalfunction, reduced renal function, or ARF. In these embodiments, theassay results, for example a measured concentration of one or moremarkers selected from the group consisting of the biomarkers listed inTable 2 herein are correlated to the occurrence or nonoccurrence of achange in renal status. The following are preferred monitoringembodiments.

In preferred monitoring embodiments, these methods comprise monitoringrenal status in a subject suffering from an injury to renal function,and the assay result(s) is/are correlated to the occurrence ornonoccurrence of a change in renal status in the subject. For example,the measured concentration(s) may be compared to a threshold value. Fora positive going marker, when the measured concentration is above thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is below the threshold,an improvement of renal function may be assigned to the subject. For anegative going marker, when the measured concentration is below thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is above the threshold,an improvement of renal function may be assigned to the subject.

In other preferred monitoring embodiments, these methods comprisemonitoring renal status in a subject suffering from reduced renalfunction, and the assay result(s) is/are correlated to the occurrence ornonoccurrence of a change in renal status in the subject. For example,the measured concentration(s) may be compared to a threshold value. Fora positive going marker, when the measured concentration is above thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is below the threshold,an improvement of renal function may be assigned to the subject. For anegative going marker, when the measured concentration is below thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is above the threshold,an improvement of renal function may be assigned to the subject.

In yet other preferred monitoring embodiments, these methods comprisemonitoring renal status in a subject suffering from acute renal failure,and the assay result(s) is/are correlated to the occurrence ornonoccurrence of a change in renal status in the subject. For example,the measured concentration(s) may be compared to a threshold value. Fora positive going marker, when the measured concentration is above thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is below the threshold,an improvement of renal function may be assigned to the subject. For anegative going marker, when the measured concentration is below thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is above the threshold,an improvement of renal function may be assigned to the subject.

In other additional preferred monitoring embodiments, these methodscomprise monitoring renal status in a subject at risk of an injury torenal function due to the pre-existence of one or more known riskfactors for prerenal, intrinsic renal, or postrenal ARF, and the assayresult(s) is/are correlated to the occurrence or nonoccurrence of achange in renal status in the subject. For example, the measuredconcentration(s) may be compared to a threshold value. For a positivegoing marker, when the measured concentration is above the threshold, aworsening of renal function may be assigned to the subject;alternatively, when the measured concentration is below the threshold,an improvement of renal function may be assigned to the subject. For anegative going marker, when the measured concentration is below thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is above the threshold,an improvement of renal function may be assigned to the subject.

In still other embodiments, the methods for evaluating renal statusdescribed herein are methods for classifying a renal injury in thesubject; that is, determining whether a renal injury in a subject isprerenal, intrinsic renal, or postrenal; and/or further subdividingthese classes into subclasses such as acute tubular injury, acuteglomerulonephritis acute tubulointerstitial nephritis, acute vascularnephropathy, or infiltrative disease; and/or assigning a likelihood thata subject will progress to a particular RIFLE stage. In theseembodiments, the assay results, for example a measured concentration ofone or more markers selected from the group consisting of the biomarkerslisted in Table 2 herein are correlated to a particular class and/orsubclass. The following are preferred classification embodiments.

In preferred classification embodiments, these methods comprisedetermining whether a renal injury in a subject is prerenal, intrinsicrenal, or postrenal; and/or further subdividing these classes intosubclasses such as acute tubular injury, acute glomerulonephritis acutetubulointerstitial nephritis, acute vascular nephropathy, orinfiltrative disease; and/or assigning a likelihood that a subject willprogress to a particular RIFLE stage, and the assay result(s) is/arecorrelated to the injury classification for the subject. For example,the measured concentration may be compared to a threshold value, andwhen the measured concentration is above the threshold, a particularclassification is assigned; alternatively, when the measuredconcentration is below the threshold, a different classification may beassigned to the subject.

A variety of methods may be used by the skilled artisan to arrive at adesired threshold value for use in these methods. For example, thethreshold value may be determined from a population of normal subjectsby selecting a concentration representing the 75^(th), 85^(th), 90^(th),95^(th), or 99^(th) percentile of a kidney injury marker measured insuch normal subjects. Alternatively, the threshold value may bedetermined from a “diseased” population of subjects, e.g., thosesuffering from an injury or having a predisposition for an injury (e.g.,progression to ARF or some other clinical outcome such as death,dialysis, renal transplantation, etc.), by selecting a concentrationrepresenting the 75^(th), 85^(th), 90^(th), 95^(th), or 99^(th)percentile of a kidney injury marker measured in such subjects. Inanother alternative, the threshold value may be determined from a priormeasurement of a kidney injury marker in the same subject; that is, atemporal change in the level of a kidney injury marker in the subjectmay be used to assign risk to the subject.

The foregoing discussion is not meant to imply, however, that the kidneyinjury markers of the present invention must be compared tocorresponding individual thresholds. Methods for combining assay resultscan comprise the use of multivariate logistical regression, loglinearmodeling, neural network analysis, n-of-m analysis, decision treeanalysis, calculating ratios of markers, etc. This list is not meant tobe limiting. In these methods, a composite result which is determined bycombining individual markers may be treated as if it is itself a marker;that is, a threshold may be determined for the composite result asdescribed herein for individual markers, and the composite result for anindividual patient compared to this threshold.

The ability of a particular test or combination of tests to distinguishtwo populations can be established using ROC analysis. For example, ROCcurves established from a “first” subpopulation which is predisposed toone or more future changes in renal status, and a “second” subpopulationwhich is not so predisposed can be used to calculate a ROC curve, andthe area under the curve provides a measure of the quality of the test.Preferably, the tests described herein provide a ROC curve area greaterthan 0.5, preferably at least 0.6, more preferably 0.7, still morepreferably at least 0.8, even more preferably at least 0.9, and mostpreferably at least 0.95.

In certain aspects, the measured concentration of one or more kidneyinjury markers, or a composite of such markers, may be treated ascontinuous variables. For example, any particular concentration can beconverted into a corresponding probability of a future reduction inrenal function for the subject, the occurrence of an injury, aclassification, etc. In yet another alternative, a threshold that canprovide an acceptable level of specificity and sensitivity in separatinga population of subjects into “bins” such as a “first” subpopulation(e.g., which is predisposed to one or more future changes in renalstatus, the occurrence of an injury, a classification, etc.) and a“second” subpopulation which is not so predisposed. A threshold value isselected to separate this first and second population by one or more ofthe following measures of test accuracy:

-   an odds ratio greater than 1, preferably at least about 2 or more or    about 0.5 or less, more preferably at least about 3 or more or about    0.33 or less, still more preferably at least about 4 or more or    about 0.25 or less, even more preferably at least about 5 or more or    about 0.2 or less, and most preferably at least about 10 or more or    about 0.1 or less;-   a specificity of greater than 0.5, preferably at least about 0.6,    more preferably at least about 0.7, still more preferably at least    about 0.8, even more preferably at least about 0.9 and most    preferably at least about 0.95, with a corresponding sensitivity    greater than 0.2, preferably greater than about 0.3, more preferably    greater than about 0.4, still more preferably at least about 0.5,    even more preferably about 0.6, yet more preferably greater than    about 0.7, still more preferably greater than about 0.8, more    preferably greater than about 0.9, and most preferably greater than    about 0.95;-   a sensitivity of greater than 0.5, preferably at least about 0.6,    more preferably at least about 0.7, still more preferably at least    about 0.8, even more preferably at least about 0.9 and most    preferably at least about 0.95, with a corresponding specificity    greater than 0.2, preferably greater than about 0.3, more preferably    greater than about 0.4, still more preferably at least about 0.5,    even more preferably about 0.6, yet more preferably greater than    about 0.7, still more preferably greater than about 0.8, more    preferably greater than about 0.9, and most preferably greater than    about 0.95;-   at least about 75% sensitivity, combined with at least about 75%    specificity;-   a positive likelihood ratio (calculated as    sensitivity/(1−specificity)) of greater than 1, at least about 2,    more preferably at least about 3, still more preferably at least    about 5, and most preferably at least about 10; or-   a negative likelihood ratio (calculated as    (1−sensitivity)/specificity) of less than 1, less than or equal to    about 0.5, more preferably less than or equal to about 0.3, and most    preferably less than or equal to about 0.1.-   The term “about” in the context of any of the above measurements    refers to +/−5% of a given measurement.

Multiple thresholds may also be used to assess renal status in asubject. For example, a “first” subpopulation which is predisposed toone or more future changes in renal status, the occurrence of an injury,a classification, etc., and a “second” subpopulation which is not sopredisposed can be combined into a single group. This group is thensubdivided into three or more equal parts (known as tertiles, quartiles,quintiles, etc., depending on the number of subdivisions). An odds ratiois assigned to subjects based on which subdivision they fall into. Ifone considers a tertile, the lowest or highest tertile can be used as areference for comparison of the other subdivisions. This referencesubdivision is assigned an odds ratio of 1. The second tertile isassigned an odds ratio that is relative to that first tertile. That is,someone in the second tertile might be 3 times more likely to suffer oneor more future changes in renal status in comparison to someone in thefirst tertile. The third tertile is also assigned an odds ratio that isrelative to that first tertile.

In certain embodiments, the assay method is an immunoassay. Antibodiesfor use in such assays will specifically bind a full length kidneyinjury marker of interest, and may also bind one or more polypeptidesthat are “related” thereto, as that term is defined hereinafter.Numerous immunoassay formats are known to those of skill in the art.Preferred body fluid samples are selected from the group consisting ofurine, blood, serum, saliva, tears, and plasma.

As previously noted, preferred biomarker panels comprise two, three,four, five, or more assays configured to detect two, three, four, five,or more of the biomarkers listed in Table 2 herein. That said, theforegoing method steps should not be interpreted to mean that the kidneyinjury marker assay result(s) is/are used in isolation in the methodsdescribed herein. Rather, additional variables or other clinical indiciamay be included in the methods described herein. For example, a riskstratification, diagnostic, classification, monitoring, etc. method maycombine the assay result(s) with one or more variables measured for thesubject selected from the group consisting of demographic information(e.g., weight, sex, age, race), medical history (e.g., family history,type of surgery, pre-existing disease such as aneurism, congestive heartfailure, preeclampsia, eclampsia, diabetes mellitus, hypertension,coronary artery disease, proteinuria, renal insufficiency, or sepsis,type of toxin exposure such as NSAIDs, cyclosporines, tacrolimus,aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin,ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, orstreptozotocin), clinical variables (e.g., blood pressure, temperature,respiration rate), risk scores (APACHE score, PREDICT score, TIMI RiskScore for UA/NSTEMI, Framingham Risk Score, risk scores of Thakar et al.(J. Am. Soc. Nephrol. 16: 162-68, 2005), Mehran et al. (J. Am. Coll.Cardiol. 44: 1393-99, 2004), Wijeysundera et al. (JAMA 297: 1801-9,2007), Goldstein and Chawla (Clin. J. Am. Soc. Nephrol. 5: 943-49,2010), or Chawla et al. (Kidney Intl. 68: 2274-80, 2005)), a glomerularfiltration rate, an estimated glomerular filtration rate, a urineproduction rate, a serum or plasma creatinine concentration, a urinecreatinine concentration, a fractional excretion of sodium, a urinesodium concentration, a urine creatinine to serum or plasma creatinineratio, a urine specific gravity, a urine osmolality, a urine ureanitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnine ratio,a renal failure index calculated as urine sodium/(urinecreatinine/plasma creatinine), a serum or plasma neutrophil gelatinase(NGAL) concentration, a urine NGAL concentration, a serum or plasmacystatin C concentration, a serum or plasma cardiac troponinconcentration, a serum or plasma BNP concentration, a serum or plasmaNTproBNP concentration, and a serum or plasma proBNP concentration.Other measures of renal function which may be combined with one or morekidney injury marker assay result(s) are described hereinafter and inHarrison's Principles of Internal Medicine, 17^(th) Ed., McGraw Hill,N.Y., pages 1741-1830, and Current Medical Diagnosis & Treatment 2008,47^(th) Ed, McGraw Hill, New York, pages 785-815, each of which arehereby incorporated by reference in their entirety.

When more than one marker is measured, the individual markers may bemeasured in samples obtained at the same time, or may be determined fromsamples obtained at different (e.g., an earlier or later) times. Theindividual markers may also be measured on the same or different bodyfluid samples. For example, one kidney injury marker may be measured ina serum or plasma sample and another kidney injury marker may bemeasured in a urine sample. In addition, assignment of a likelihood maycombine an individual kidney injury marker assay result with temporalchanges in one or more additional variables.

In various related aspects, the present invention also relates todevices and kits for performing the methods described herein. Suitablekits comprise reagents sufficient for performing an assay for at leastone of the described kidney injury markers, together with instructionsfor performing the described threshold comparisons.

In certain embodiments, reagents for performing such assays are providedin an assay device, and such assay devices may be included in such akit. Preferred reagents can comprise one or more solid phase antibodies,the solid phase antibody comprising antibody that detects the intendedbiomarker target(s) bound to a solid support. In the case of sandwichimmunoassays, such reagents can also include one or more detectablylabeled antibodies, the detectably labeled antibody comprising antibodythat detects the intended biomarker target(s) bound to a detectablelabel. Additional optional elements that may be provided as part of anassay device are described hereinafter.

Detectable labels may include molecules that are themselves detectable(e.g., fluorescent moieties, electrochemical labels, ecl(electrochemical luminescence) labels, metal chelates, colloidal metalparticles, etc.) as well as molecules that may be indirectly detected byproduction of a detectable reaction product (e.g., enzymes such ashorseradish peroxidase, alkaline phosphatase, etc.) or through the useof a specific binding molecule which itself may be detectable (e.g., alabeled antibody that binds to the second antibody, biotin, digoxigenin,maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA,dsDNA, etc.).

Generation of a signal from the signal development element can beperformed using various optical, acoustical, and electrochemical methodswell known in the art. Examples of detection modes include fluorescence,radiochemical detection, reflectance, absorbance, amperometry,conductance, impedance, interferometry, ellipsometry, etc. In certain ofthese methods, the solid phase antibody is coupled to a transducer(e.g., a diffraction grating, electrochemical sensor, etc) forgeneration of a signal, while in others, a signal is generated by atransducer that is spatially separate from the solid phase antibody(e.g., a fluorometer that employs an excitation light source and anoptical detector). This list is not meant to be limiting. Antibody-basedbiosensors may also be employed to determine the presence or amount ofanalytes that optionally eliminate the need for a labeled molecule.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-43 provide a detailed summary of the ability of panels ofbiomarkers to evaluate acute kidney injury in an ICU patient population.Patients in the population are divided based on the the RIFLEclassification of renal status. The following analyses are provided:

FIG. 1. No or R RIFLE stage versus RIFLE I or F. RIFLE stage adjudicatedby sCr and Urine Output. Diseased group sample is obtained 24 hoursprior to RIFLE I diagnosis.

FIG. 2. No or R RIFLE stage versus RIFLE I or F. RIFLE stage adjudicatedby sCr. Diseased group sample is obtained 24 hours prior to RIFLE Idiagnosis.

FIG. 3. No or R RIFLE stage versus RIFLE I or F. RIFLE stage adjudicatedby Urine Output. Diseased group sample is obtained 24 hours prior toRIFLE I diagnosis.

FIG. 4. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage adjudicatedby sCr and Urine Output. Diseased group sample is obtained 24 hoursprior to RIFLE R diagnosis.

FIG. 5. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage adjudicatedby sCr. Diseased group sample is obtained 24 hours prior to RIFLE Rdiagnosis.

FIG. 6. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage adjudicatedby Urine Output. Diseased group sample is obtained 24 hours prior toRIFLE R diagnosis.

FIG. 7. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage adjudicatedby sCr and Urine Output. Diseased group sample is obtained 24 hoursprior to RIFLE I diagnosis.

FIG. 8. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage adjudicatedby Urine Output. Diseased group sample is obtained 24 hours prior toRIFLE I diagnosis.

FIG. 9. No or R RIFLE stage versus RIFLE I or F. RIFLE stage adjudicatedby sCr and Urine Output. Diseased group sample is obtained at RIFLE Idiagnosis.

FIG. 10. No or R RIFLE stage versus RIFLE I or F. RIFLE stageadjudicated by sCr. Diseased group sample is obtained at RIFLE Idiagnosis.

FIG. 11. No or R RIFLE stage versus RIFLE I or F. RIFLE stageadjudicated by Urine Output. Diseased group sample is obtained at RIFLEI diagnosis.

FIG. 12. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage adjudicatedby sCr and Urine Output. Diseased group sample is obtained at RIFLE Rdiagnosis.

FIG. 13. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage adjudicatedby sCr. Diseased group sample is obtained at RIFLE R diagnosis.

FIG. 14. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage adjudicatedby Urine Output. Diseased group sample is obtained at RIFLE R diagnosis.

FIG. 15. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage adjudicatedby sCr and Urine Output. Diseased group sample is obtained at RIFLE Idiagnosis.

FIG. 16. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage adjudicatedby sCr. Diseased group sample is obtained at RIFLE I diagnosis.

FIG. 17. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage adjudicatedby Urine Output. Diseased group sample is obtained at RIFLE I diagnosis.

FIG. 18. No or R RIFLE stage versus RIFLE I or F. RIFLE stageadjudicated by sCr and Urine Output. Diseased group sample is obtained48 hours prior to RIFLE I diagnosis.

FIG. 19. No or R RIFLE stage versus RIFLE I or F. RIFLE stageadjudicated by sCr. Diseased group sample is obtained 48 hours prior toRIFLE I diagnosis.

FIG. 20. No or R RIFLE stage versus RIFLE I or F. RIFLE stageadjudicated by Urine Output. Diseased group sample is obtained 48 hoursprior to RIFLE I diagnosis.

FIG. 21. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage adjudicatedby sCr and Urine Output. Diseased group sample is obtained 48 hoursprior to RIFLE R diagnosis.

FIG. 22. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage adjudicatedby sCr. Diseased group sample is obtained 48 hours prior to RIFLE Rdiagnosis.

FIG. 23. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage adjudicatedby Urine Output. Diseased group sample is obtained 48 hours prior toRIFLE R diagnosis.

FIG. 24. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage adjudicatedby sCr and Urine Output. Diseased group sample is obtained 48 hoursprior to RIFLE I diagnosis.

FIG. 25. Progression of RIFLE R to RIFLE I or F. RIFLE stage adjudicatedby sCr and Urine Output. Sample is obtained at R diagnosis.

FIG. 26. Progression of RIFLE R to RIFLE I or F. RIFLE stage adjudicatedby sCr. Sample is obtained at R diagnosis.

FIG. 27. Progression of RIFLE R to RIFLE I or F. RIFLE stage adjudicatedby Urine Output. Sample is obtained at R diagnosis.

FIG. 28. No or R RIFLE stage versus RIFLE I or F. RIFLE stageadjudicated by sCr and Urine Output, within 48 hrs of enrollment. Sampleis obtained at enrollment.

FIG. 29. No or R RIFLE stage versus RIFLE I or F. RIFLE stageadjudicated by sCr, within 48 hrs of enrollment. Sample is obtained atenrollment.

FIG. 30. No or R RIFLE stage versus RIFLE I or F. RIFLE stageadjudicated by Urine Output, within 48 hrs of enrollment. Sample isobtained at enrollment.

FIG. 31. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage adjudicatedby sCr and Urine Output, within 48 hrs of enrollment. Sample is obtainedat enrollment.

FIG. 32. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage adjudicatedby sCr, within 48 hrs of enrollment. Sample is obtained at enrollment.

FIG. 33. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage adjudicatedby Urine Output, within 48 hrs of enrollment. Sample is obtained atenrollment.

FIG. 34. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage adjudicatedby sCr and Urine Output, within 48 hrs of enrollment. Sample is obtainedat enrollment.

FIG. 35. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage adjudicatedby Urine Output, within 48 hrs of enrollment. Sample is obtained atenrollment.

FIG. 36. No or R RIFLE stage versus RIFLE I or F. RIFLE stageadjudicated by sCr and Urine Output, within 24 hrs of enrollment. Sampleis obtained at enrollment.

FIG. 37. No or R RIFLE stage versus RIFLE I or F. RIFLE stageadjudicated by sCr, within 24 hrs of enrollment. Sample is obtained atenrollment.

FIG. 38. No or R RIFLE stage versus RIFLE I or F. RIFLE stageadjudicated by Urine Output, within 24 hrs of enrollment. Sample isobtained at enrollment.

FIG. 39. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage adjudicatedby sCr and Urine Output, within 24 hrs of enrollment. Sample is obtainedat enrollment.

FIG. 40. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage adjudicatedby sCr, within 24 hrs of enrollment. Sample is obtained at enrollment.

FIG. 41. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage adjudicatedby Urine Output, within 24 hrs of enrollment. Sample is obtained atenrollment.

FIG. 42. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage adjudicatedby sCr and Urine Output, within 24 hrs of enrollment. Sample is obtainedat enrollment.

FIG. 43. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage adjudicatedby Urine Output, within 24 hrs of enrollment. Sample is obtained atenrollment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for diagnosis,differential diagnosis, risk stratification, monitoring, classifying anddetermination of treatment regimens in subjects suffering or at risk ofsuffering from injury to renal function, reduced renal function and/oracute renal failure through measurement of one or more kidney injurymarkers. In various embodiments, a biomarker recited in Table 2 herein,or one or more markers related thereto, is combined with one or moreadditional biomarkers listed in Table 2 herein and/or with one or moreother biomarkers or clinical indicia, and the combination correlated tothe renal status of the subject.

For purposes of this document, the following definitions apply:

As used herein, an “injury to renal function” is an abrupt (within 14days, preferably within 7 days, more preferably within 72 hours, andstill more preferably within 48 hours) measurable reduction in a measureof renal function. Such an injury may be identified, for example, by adecrease in glomerular filtration rate or estimated GFR, a reduction inurine output, an increase in serum creatinine, an increase in serumcystatin C, a requirement for renal replacement therapy, etc.“Improvement in Renal Function” is an abrupt (within 14 days, preferablywithin 7 days, more preferably within 72 hours, and still morepreferably within 48 hours) measurable increase in a measure of renalfunction. Preferred methods for measuring and/or estimating GFR aredescribed hereinafter.

As used herein, “reduced renal function” is an abrupt (within 14 days,preferably within 7 days, more preferably within 72 hours, and stillmore preferably within 48 hours) reduction in kidney function identifiedby an absolute increase in serum creatinine of greater than or equal to0.1 mg/dL (≧8.8 μmol/L), a percentage increase in serum creatinine ofgreater than or equal to 20% (1.2-fold from baseline), or a reduction inurine output (documented oliguria of less than 0. 5 ml/kg per hour).

As used herein, “acute renal failure” or “ARF” is an abrupt (within 14days, preferably within 7 days, more preferably within 72 hours, andstill more preferably within 48 hours) reduction in kidney functionidentified by an absolute increase in serum creatinine of greater thanor equal to 0.3 mg/dl (≧26.4 μmol/L), a percentage increase in serumcreatinine of greater than or equal to 50% (1. 5-fold from baseline), ora reduction in urine output (documented oliguria of less than 0.5 ml/kgper hour for at least 6 hours). This term is synonymous with “acutekidney injury” or “AKI.”

In the case of polypeptide biomarkers used herein, a “marker” or“biomarker” refers to polypeptides present in a biological sample thatare derived from a particular biosynthetic precursor. In the case ofbiomarkers that are not polypeptides (e.g., Hyaluronic acid), a “marker”or “biomarker” refers to the particular molecular entity recited.

The following Table 2 provides a list of preferred biomarkers findinguse in the present invention. In the table, the “recommended name” forthe biomarker precursor from the Swiss-Prot “UniProtKB” database, andfor most polypeptide biomarkers the Swiss-Prot entry number for thehuman precursor. In the event that the assay detects a complex, theSwiss Prot entry is listed for each member of the complex.

TABLE 2 Preferred Name Swiss-Prot: Preferred Name Swiss-Prot: 60 kDaheat shock protein, P10809 72 kDa type IV collagenase P08253mitochondrial 72 kDa type IV P08253 72 kDa type IV P08253 P16035collagenase: Metalloproteinase P01033 collagenase: Metalloproteinaseinhibitor 1 complex inhibitor 2 complex 72 kDa type IV P08253Adiponectin Q15848 collagenase: Metalloproteinase Q99727 inhibitor 4complex Advanced glycosylation end Q15109 Agouti-related protein O00253product-specific receptor Alkaline phosphatase, tissue- P05186Alpha-1-antitrypsin P01009 nonspecific isozyme Alpha-1-antitrypsin:Neutrophil P01009 Alpha-1- P01009 P00747 elastase complex P08246antitrypsin: Plasminogen complex Alpha-2 macroglobulin P01023Alpha-2-HS-glycoprotein P02765 Alpha-fetoprotein P02771 AmphiregulinP15514 Amyloid Beta 40 P05067 Amyloid Beta 42 P05067 (aa672- (aa672-711)713) Angiogenin P03950 Angiopoietin-1 Q15389 Angiopoietin-1 receptorQ02763 Angiopoietin-2 O15123 Angiopoietin-related protein 3 Q9Y5C1Angiopoietin-related protein 4 Q9BY76 Angiopoietin-related protein 6Q8NI99 Anti-Cathepsin-G (ANCA) NA Antileukoproteinase P03973Apolipoprotein A-I P02647 Apolipoprotein A-II P02652 ApolipoproteinB-100 P04114 Apolipoprotein C-III P02656 Apolipoprotein E P02649Apolipoprotein(a) P08519 Appetite-regulating hormone Q9UBU3 Aspartateaminotransferase, P17174 Bactericidal permeability- P17213 cytoplasmicincreasing protein Bc12 antagonist of cell death Q92934Beta-2-glycoprotein 1 P02749 Beta-2-microglobulin P61769 Beta-nervegrowth factor P01138 Betacellulin P35070 Bone morphogenetic protein 7P18075 Brain-derived neurotrophic factor P23560 C-C motif chemokine 1P22362 C-C motif chemokine 13 Q99616 C-C motif chemokine 15 Q16663 C-Cmotif chemokine 17 Q92583 C-C motif chemokine 18 P55774 C-C motifchemokine 19 Q99731 C-C motif chemokine 2 P13500 C-C motif chemokine 20P78556 C-C motif chemokine 21 O00585 C-C motif chemokine 22 O00626 C-Cmotif chemokine 23 P55773 C-C motif chemokine 24 O00175 C-C motifchemokine 26 Q9Y258 C-C motif chemokine 27 Q9Y4X3 C-C motif chemokine 3P10147 C-C motif chemokine 4 P13236 C-C motif chemokine 5 P13501 C-Cmotif chemokine 7 P80098 C-C motif chemokine 8 P80075 C-PeptideP01308(aa5 C-reactive protein P02741 7-87) C-X-C motif chemokine 10P02778 C-X-C motif chemokine 11 O14625 C-X-C motif chemokine 13 O43927C-X-C motif chemokine 16 Q9H2A7 C-X-C motif chemokine 2 P19875 C-X-Cmotif chemokine 5 P42830 C-X-C motif chemokine 6 P80162 C-X-C motifchemokine 9 Q07325 Cadherin-1 P12830 Cadherin-16 O75309 Cadherin-3P22223 Cadherin-5 P33151 Calbindin P05937 Calcitonin P01258 Calcitonin(Procalcitonin) P01258-Pro Cancer Antigen 15-3 NA Cancer Antigen 19-9 NACarbonic anhydrase 9 Q16790 Carcinoembryonic antigen-related P13688Carcinoembryonic antigen- P06731 cell adhesion molecule 1 related celladhesion molecule 5 Caspase- 1 P29466 Caspase-3, active P42574 Caspase-8Q14790 Caspase-9 P55211 Cathepsin B P07858 Cathepsin D P07339 CathepsinS P25774 CD40 ligand P29965 CD44 antigen P16070 Cellular tumor antigenp53 P04637 Choriogonadotropin subunit beta P01233 Ciliary neurotrophicfactor P26441 Clusterin P10909 Coagulation factor VII P08709 Collagenase3 P45452 Complement C3 P01024 Complement C4-B P0C0L5 Complement C5P01031 Complement factor H P08603 Corticotropin P01189(aa138- 176)Cortisol NA Creatine Kinase-MB P12277 P06732 Creatinine NACyclin-dependent kinase P38936 inhibitor 1 Cystatin-C P01034 Cytochromec P99999 DDRGK domain-containing Q96HY6 Dipeptidyl peptidase 4 P27487protein 1 E-selectin P16581 Endoglin P17813 Endostatin P39060(aa1Endothelial protein C receptor Q9UNN8 572-1754) Endothelin-1 P05305Eotaxin P51671 Epidermal growth factor receptor P00533 Epiregulin O14944Epithelial cell adhesion molecule P16422 Erythropoietin P01588Erythropoietin receptor P19235 Fatty acid-binding protein, P05413 heartFatty acid-binding protein, P12104 Fatty acid-binding protein, P07148intestinal liver Ferritin P02792 Fibrinogen P02671 P02675 P02794 P02679Fibroblast growth factor 19 O95750 Fibroblast growth factor 21 Q9NSA1Fibroblast growth factor 23 Q9GZV9 Fibronectin P02751 Follistatin P19883Follitropin P01215 P01225 Follitropin subunit beta P01225 FractalkineP78423 Galectin-3 P17931 Gastric inhibitory polypeptide P09681 Glialcell line-derived P39905 Glial fibrillary acidic protein P14136neurotrophic factor Glucagon P01275 Glucagon-like peptide 1 P01275(aa98-127 aa98-128) Glutathione S-transferase A1 P08263 GlutathioneS-transferase P P09211 Granulocyte colony-stimulating P09919Granulocyte-macrophage P04141 factor colony-stimulating factor GranzymeB P10144 Granzyme M P51124 Growth-regulated alpha protein P09341Haptoglobin P00738 Heat shock 70 kDa protein 1 P08107 Heat shock proteinbeta-1 P04792 Heat shock protein beta-1 P04792 Heat shock protein HSP90- P07900 (phospho SER78 / phospho (pS78/pS82) alpha SER82) Hemeoxygenase 1 P09601 Heparan Sulfate NA Heparin-binding EGF-like growthQ99075 Heparin-binding growth factor P05230 factor 1 Heparin-bindinggrowth factor 2 P09038 Hepatitis A virus cellular O43656 receptor 1Hepatocyte growth factor P14210 Hepatocyte growth factor P08581 receptorHyaluronic acid NA Hypoxia-inducible factor 1 Q16665 alphaImmunoglobulin A NA Immunoglobulin E NA Immunoglobulin M NAImmunoglogulin G1 NA Immunoglogulin G2 NA Immunoglogulin G3 NAImmunoglogulin G4 NA Insulin P01308 Insulin receptor substrate 1 P35568Insulin-like growth factor 1 P08069 receptor Insulin-like growth factorIA P01343 Insulin-like growth factor- P08833 binding protein 1Insulin-like growth factor-binding P18065 Insulin-like growth factor-P17936 protein 2 binding protein 3 Insulin-like growth factor-bindingP22692 Insulin-like growth factor- P24593 protein 4 binding protein 5Insulin-like growth factor-binding P24592 Insulin-like growth factor-Q16270 protein 6 binding protein 7 Intercellular adhesion molecule 1P05362 Intercellular adhesion P13598 molecule 2 Intercellular adhesionmolecule 3 P32942 Interferon alpha-2 P01563 Interferon gamma P01579Interleukin-1 alpha P01583 Interleukin-1 beta P01584 Interleukin-1receptor P18510 antagonist protein Interleukin-1 receptor type I P14778Interleukin-1 receptor type II P27930 Interleukin-10 P22301 Interleukin-11 P20809 Interleukin-12 P29459 Interleukin-12 subunit beta P29460P29460 Interleukin-13 P35225 Interleukin-15 P40933 Interleukin-17AQ16552 Interleukin-18 Q14116 Interleukin-2 P60568 Interleukin-2 receptoralpha P01589 chain Interleukin-20 Q9NYY1 Interleukin-21 Q9HBE4Interleukin-23 Q9NPF7 Interleukin-28A Q8IZJ0 P29460 Interleukin-29Q8IU54 Interleukin-3 P08700 Interleukin-33 O95760 Interleukin-4 P05112Interleukin-4 receptor alpha chain P24394 Interleukin-5 P05113Interleukin-6 P05231 Interleukin-6 receptor subunit P08887 alphaInterleukin-6 receptor subunit beta P40189 Interleukin-7 P13232Interleukin-8 P10145 Interleukin-9 P15248 Interstitial collagenaseP03956 Interstitial P03956 P16035 collagenase: Metalloproteinaseinhibitor 2 complex Involucrin P07476 Islet amyloid polypeptide P10997Keratin, type I cytoskeletal 19 P08727 Keratin, type II cytoskeletal 1;P04264 P13645 (aa311-367) typel cytoskeletal 10 (Keratin- 1,-10 mix)Keratin, type II cytoskeletal 6 (6A, P02538 Kit ligand P21583 -6B, -6Cmix) P04259 P48668 Lactotransferrin P02788 Leptin P41159 Leukemiainhibitory factor P15018 Lipopolysaccharide (serotypes NA -K,-O)Lutropin P01215 Lutropin subunit beta P01229 P01229 Lymphatic vesselendothelial Q9Y5Y7 Lymphotactin P47992 hyaluronic acid receptor 1Lymphotoxin-alpha P01374 Lysozyme C P61626 Macrophage colony-stimulatingP09603 Macrophage metalloelastase P39900 factor 1 Macrophage migrationinhibitory P14174 Malondialdehyde-modified NA factor low-densitylipoprotein Matrilysin P09237 Matrix metalloproteinase-9 P14780 Matrixmetalloproteinase- P14780 Matrix metalloproteinase- P14780 P35625 9:Metalloproteinase inhibitor 2 P16035 9: Metalloproteinase inhibitor 3complex complex Metalloproteinase inhibitor 1 P01033 Metalloproteinaseinhibitor 2 P16035 Metalloproteinase inhibitor 3 P35625Metalloproteinase inhibitor 4 Q99727 Midkine P21741 Mix ofGrowth-regulated P09341 P19875 alpha, beta, and gamma P19876 proteinsMonocyte differentiation antigen P08571 Mucin-16 Q8WXI7 CD14 Myeloiddifferentiation primary Q99836 Myeloperoxidase P05164 response proteinMyD88 Myoglobin P02144 Neprilysin P08473 Netrin-1 O95631 Neural celladhesion molecule P13591 1 Neuronal cell adhesion molecule Q92823Neutrophil collagenase P22894 Neutrophil elastase P08246 Neutrophilgelatinase- P80188 associated lipocalin NF-kappa-B inhibitor alphaP25963 Nidogen-1 P14543 Nitric oxide synthase, inducible P35228NT-pro-BNP P16860 Osteocalcin P02818 Osteopontin P10451 Oxidizedlow-density lipoprotein P78380 P-selectin P16109 receptor 1 P-selectinglycoprotein ligand 1 Q14242 Pancreatic prohormone P01298 Pappalysin-1Q13219 Parathyroid hormone P01270 Peptide YY P10082 Pigmentepithelium-derived P36955 factor Placenta growth factor P49763Plasminogen activator P05121 inhibitor 1 Platelet basic protein P02775Platelet endothelial cell P16284 adhesion molecule Platelet factor 4P02776 Platelet-derived growth factor P04085 P01127 A Platelet-derivedgrowth factor P04085 Platelet-derived growth factor P01127 subunit A(dimer) subunit B (dimer) Poly [ADP-ribose] polymerase 1 P09874Pro-epidermal growth factor P01133 (cleaved) Pro-Interleukin-1 betaP01584-Pro Pro-interleukin-16 Q14005 Prolactin P01236 Prostate-specificantigen P07288 Pro static acid phosphatase P15309 Protein NOV homologP48745 Protein S 100-A12 P80511 Protein S100-B P04271 Protransforminggrowth factor P01135 Renin P00797 alpha Resistin Q9HD89 Serum albuminP02768 Serum amyloid A protein P02735 Serum amyloid P-component P02743Sex hormone-binding globulin P04278 SL cytokine P49771 SomatotropinP01241 Stromal cell-derived factor 1 P48061 Stromelysin- 1 P08254Stromelysin- P08254 P16035 1: Metalloproteinase inhibitor 2 complexStromelysin-2 P09238 Tenascin P24821 Thrombomodulin P07204Thrombopoietin P40225 Thrombospondin-1 P07996 Thrombospondin-2 P35442Thymic stromal lymphopoietin Q969D9 Thyrotropin P01215 P01222Thyroxine-binding globulin P05543 Tissue factor P13726 Tissue-typeplasminogen activator P00750 Transforming growth factor P01137 beta-1Transforming growth factor beta-2 P61812 Transforming growth factorP10600 beta-3 Transmembrane glycoprotein Q14956 Transthyretin P02766 NMBTrefoil factor 3 Q07654 Tubulointerstitial nephritis Q9UJW2 antigenTumor necrosis factor P01375 Tumor necrosis factor ligand P50591superfamily member 10 Tumor necrosis factor ligand O14788 Tumor necrosisfactor ligand P48023 superfamily member 11 superfamily member 6 Tumornecrosis factor receptor O14763 Tumor necrosis factor receptor O00300superfamily member 10B superfamily member 11B Tumor necrosis factorreceptor P19438 Tumor necrosis factor receptor P20333 superfamily member1A superfamily member 1B Tumor necrosis factor receptor P25942 Tumornecrosis factor receptor P25445 superfamily member 5 superfamily member6 Tumor necrosis factor receptor P28908 Urokinase plasminogen Q03405superfamily member 8 activator surface receptor Urokinase-typeplasminogen P00749 Vascular cell adhesion protein P19320 activator 1Vascular endothelial growth factor P15692 Vascular endothelial growth043915 A factor D Vascular endothelial growth factor P17948 Vascularendothelial growth P35968 receptor 1 factor receptor 2 Vascularendothelial growth factor P35916 Versican core protein P13611 receptor 3Vitamin D-binding protein P02774 Vitamin K-dependent protein P04070 Cvon Willebrand Factor P04275 WAP four-disulfide core Q14508 domainprotein 2

Included in this list are a number of proteins which exist in one formas type-I, type-II, or GPI-anchored membrane proteins. Typically, suchmembrane proteins comprise a substantial extracellular domain, some orall of which can be detected as soluble forms present in aqueous samplessuch as blood, serum, plasma, urine, etc., either as cleavage productsor as splice variants which delete an effective membrane spanningdomain. These membrane proteins include Swiss-Prot entry numbers O14788,O14944, O75309, P00797, P05186, P08473, P13688, P15514, P22223, P27487,P35070, Q03405, Q14956, Q16790, Q99075, Q9Y5Y7Q15109, Q02763, P17213,P12830, P33151, P06731, P29965, P16070, Q9H2A7, P17813, Q9UNN8, P00533,P16422, P19235, P16581, P78423, O43656, P08581, P08069, P05362, P13598,P32942, P14778, P27930, P01589, P24394, P08887, P40189, P21583, P09603,P08571, Q8WXI7, P13591, Q92823, P78380, P16284, P01133, P15309, P01135,P16109, Q14242, P49771, P07204, P13726, P01375, P50591, P48023, 014763,P19438, P20333, P25942, P25445, P28908, P19320, P17948, and P35968.Preferred assays detect soluble forms of these biomarkers.

In this regard, the skilled artisan will understand that the signalsobtained from an immunoassay are a direct result of complexes formedbetween one or more antibodies and the corresponding target biomolecule(i.e., the analyte) containing the necessary epitope(s) to which theantibodies bind. While such assays may detect the full length biomarkerand the assay result be expressed as a concentration of a biomarker ofinterest, the signal from the assay is actually a result of all such“immunoreactive” molecules present in the sample. Expression ofbiomarkers may also be determined by means other than immunoassays,including protein measurements (such as dot blots, western blots,chromatographic methods, mass spectrometry, etc.) and nucleic acidmeasurements (mRNA quatitation). This list is not meant to be limiting.

As used herein, the term “relating a signal to the presence or amount”of an analyte reflects this understanding. Assay signals are typicallyrelated to the presence or amount of an analyte through the use of astandard curve calculated using known concentrations of the analyte ofinterest. As the term is used herein, an assay is “configured to detect”an analyte if an assay can generate a detectable signal indicative ofthe presence or amount of a physiologically relevant concentration ofthe analyte. Because an antibody epitope is on the order of 8 aminoacids, an immunoassay configured to detect a marker of interest willalso detect polypeptides related to the marker sequence, so long asthose polypeptides contain the epitope(s) necessary to bind to theantibody or antibodies used in the assay. The term “related marker” asused herein with regard to a biomarker such as one of the kidney injurymarkers described herein refers to one or more fragments, variants,etc., of a particular marker or its biosynthetic parent that may bedetected as a surrogate for the marker itself or as independentbiomarkers. The term also refers to one or more polypeptides present ina biological sample that are derived from the biomarker precursorcomplexed to additional species, such as binding proteins, receptors,heparin, lipids, sugars, etc.

The term “positive going” marker as that term is used herein refer to amarker that is determined to be elevated in subjects suffering from adisease or condition, relative to subjects not suffering from thatdisease or condition. The term “negative going” marker as that term isused herein refer to a marker that is determined to be reduced insubjects suffering from a disease or condition, relative to subjects notsuffering from that disease or condition.

The term “subject” as used herein refers to a human or non-humanorganism. Thus, the methods and compositions described herein areapplicable to both human and veterinary disease. Further, while asubject is preferably a living organism, the invention described hereinmay be used in post-mortem analysis as well. Preferred subjects arehumans, and most preferably “patients,” which as used herein refers toliving humans that are receiving medical care for a disease orcondition. This includes persons with no defined illness who are beinginvestigated for signs of pathology.

Preferably, an analyte is measured in a sample. Such a sample may beobtained from a subject, or may be obtained from biological materialsintended to be provided to the subject. For example, a sample may beobtained from a kidney being evaluated for possible transplantation intoa subject, and an analyte measurement used to evaluate the kidney forpreexisting damage. Preferred samples are body fluid samples.

The term “body fluid sample” as used herein refers to a sample of bodilyfluid obtained for the purpose of diagnosis, prognosis, classificationor evaluation of a subject of interest, such as a patient or transplantdonor. In certain embodiments, such a sample may be obtained for thepurpose of determining the outcome of an ongoing condition or the effectof a treatment regimen on a condition. Preferred body fluid samplesinclude blood, serum, plasma, cerebrospinal fluid, urine, saliva,sputum, and pleural effusions. In addition, one of skill in the artwould realize that certain body fluid samples would be more readilyanalyzed following a fractionation or purification procedure, forexample, separation of whole blood into serum or plasma components.

The term “diagnosis” as used herein refers to methods by which theskilled artisan can estimate and/or determine the probability (“alikelihood”) of whether or not a patient is suffering from a givendisease or condition. In the case of the present invention, “diagnosis”includes using the results of an assay, most preferably an immunoassay,for a kidney injury marker of the present invention, optionally togetherwith other clinical characteristics, to arrive at a diagnosis (that is,the occurrence or nonoccurrence) of an acute renal injury or ARF for thesubject from which a sample was obtained and assayed. That such adiagnosis is “determined” is not meant to imply that the diagnosis is100% accurate. Many biomarkers are indicative of multiple conditions.The skilled clinician does not use biomarker results in an informationalvacuum, but rather test results are used together with other clinicalindicia to arrive at a diagnosis. Thus, a measured biomarker level onone side of a predetermined diagnostic threshold indicates a greaterlikelihood of the occurrence of disease in the subject relative to ameasured level on the other side of the predetermined diagnosticthreshold.

Similarly, a prognostic risk signals a probability (“a likelihood”) thata given course or outcome will occur. A level or a change in level of aprognostic indicator, which in turn is associated with an increasedprobability of morbidity (e.g., worsening renal function, future ARF, ordeath) is referred to as being “indicative of an increased likelihood”of an adverse outcome in a patient.

As used herein, a “plurality” as used herein refers to at least two.Preferably, a plurality refers to at least 3, more preferably at least4, even more preferably at least 5, even more preferably at least 10,and most preferably at least 20.

Marker Assays

In general, immunoassays involve contacting a sample containing orsuspected of containing a biomarker of interest with at least oneantibody that specifically binds to the biomarker. A signal is thengenerated indicative of the presence or amount of complexes formed bythe binding of polypeptides in the sample to the antibody. The signal isthen related to the presence or amount of the biomarker in the sample.Numerous methods and devices are well known to the skilled artisan forthe detection and analysis of biomarkers. See, e.g., U.S. Pat. Nos.6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272;5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and5,480,792, and The Immunoassay Handbook, David Wild, ed. Stockton Press,New York, 1994, each of which is hereby incorporated by reference in itsentirety, including all tables, figures and claims.

The assay devices and methods known in the art can utilize labeledmolecules in various sandwich, competitive, or non-competitive assayformats, to generate a signal that is related to the presence or amountof the biomarker of interest. Suitable assay formats also includechromatographic, mass spectrographic, and protein “blotting” methods.Additionally, certain methods and devices, such as biosensors andoptical immunoassays, may be employed to determine the presence oramount of analytes without the need for a labeled molecule. See, e.g.,U.S. Pat. Nos. 5,631,171; and 5,955,377, each of which is herebyincorporated by reference in its entirety, including all tables, figuresand claims. One skilled in the art also recognizes that roboticinstrumentation including but not limited to Beckman ACCESS®, AbbottAXSYM®, Roche ELECSYS®, Dade Behring STRATUS® systems are among theimmunoassay analyzers that are capable of performing immunoassays. Butany suitable immunoassay may be utilized, for example, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive bindingassays, and the like.

Antibodies or other polypeptides may be immobilized onto a variety ofsolid supports for use in assays. Solid phases that may be used toimmobilize specific binding members include include those developedand/or used as solid phases in solid phase binding assays. Examples ofsuitable solid phases include membrane filters, cellulose-based papers,beads (including polymeric, latex and paramagnetic particles), glass,silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGAgels, SPOCC gels, and multiple-well plates. An assay strip could beprepared by coating the antibody or a plurality of antibodies in anarray on solid support. This strip could then be dipped into the testsample and then processed quickly through washes and detection steps togenerate a measurable signal, such as a colored spot. Antibodies orother polypeptides may be bound to specific zones of assay deviceseither by conjugating directly to an assay device surface, or byindirect binding. In an example of the later case, antibodies or otherpolypeptides may be immobilized on particles or other solid supports,and that solid support immobilized to the device surface.

Biological assays require methods for detection, and one of the mostcommon methods for quantitation of results is to conjugate a detectablelabel to a protein or nucleic acid that has affinity for one of thecomponents in the biological system being studied. Detectable labels mayinclude molecules that are themselves detectable (e.g., fluorescentmoieties, electrochemical labels, metal chelates, etc.) as well asmolecules that may be indirectly detected by production of a detectablereaction product (e.g., enzymes such as horseradish peroxidase, alkalinephosphatase, etc.) or by a specific binding molecule which itself may bedetectable (e.g., biotin, digoxigenin, maltose, oligohistidine,2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Preparation of solid phases and detectable label conjugates oftencomprise the use of chemical cross-linkers. Cross-linking reagentscontain at least two reactive groups, and are divided generally intohomofunctional cross-linkers (containing identical reactive groups) andheterofunctional cross-linkers (containing non-identical reactivegroups). Homobifunctional cross-linkers that couple through amines,sulfhydryls or react non-specifically are available from many commercialsources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyldisulfides are thiol reactive groups. Maleimides, alkyl and arylhalides, and alpha-haloacyls react with sulfhydryls to form thiol etherbonds, while pyridyl disulfides react with sulfhydryls to produce mixeddisulfides. The pyridyl disulfide product is cleavable. Imidoesters arealso very useful for protein-protein cross-links. A variety ofheterobifunctional cross-linkers, each combining different attributesfor successful conjugation, are commercially available.

In certain aspects, the present invention provides kits for the analysisof the described kidney injury markers. The kit comprises reagents forthe analysis of at least one test sample which comprise at least oneantibody that a kidney injury marker. The kit can also include devicesand instructions for performing one or more of the diagnostic and/orprognostic correlations described herein. Preferred kits will comprisean antibody pair for performing a sandwich assay, or a labeled speciesfor performing a competitive assay, for the analyte. Preferably, anantibody pair comprises a first antibody conjugated to a solid phase anda second antibody conjugated to a detectable label, wherein each of thefirst and second antibodies that bind a kidney injury marker. Mostpreferably each of the antibodies are monoclonal antibodies. Theinstructions for use of the kit and performing the correlations can bein the form of labeling, which refers to any written or recordedmaterial that is attached to, or otherwise accompanies a kit at any timeduring its manufacture, transport, sale or use. For example, the termlabeling encompasses advertising leaflets and brochures, packagingmaterials, instructions, audio or video cassettes, computer discs, aswell as writing imprinted directly on kits.

Antibodies

The term “antibody” as used herein refers to a peptide or polypeptidederived from, modeled after or substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof,capable of specifically binding an antigen or epitope. See, e.g.Fundamental Immunology, 3rd Edition, W. E. Paul, ed., Raven Press, N.Y.(1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J.Biochem. Biophys. Methods 25:85-97. The term antibody includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”

Antibodies used in the immunoassays described herein preferablyspecifically bind to a kidney injury marker of the present invention.The term “specifically binds” is not intended to indicate that anantibody binds exclusively to its intended target since, as noted above,an antibody binds to any polypeptide displaying the epitope(s) to whichthe antibody binds. Rather, an antibody “specifically binds” if itsaffinity for its intended target is about 5-fold greater when comparedto its affinity for a non-target molecule which does not display theappropriate epitope(s). Preferably the affinity of the antibody will beat least about 5 fold, preferably 10 fold, more preferably 25-fold, evenmore preferably 50-fold, and most preferably 100-fold or more, greaterfor a target molecule than its affinity for a non-target molecule. Inpreferred embodiments, Preferred antibodies bind with affinities of atleast about 10⁷ M⁻¹, and preferably between about 10⁸ M-1 to about 10⁹M⁻¹, about 10⁹ M⁻¹ to about 10¹⁰ M⁻¹, or about 10¹⁰ M⁻¹ to about 10¹²M⁻¹.

Affinity is calculated as Kd=koff/kon (koff is the dissociation rateconstant, Kon is the association rate constant and Kd is the equilibriumconstant). Affinity can be determined at equilibrium by measuring thefraction bound (r) of labeled ligand at various concentrations (c). Thedata are graphed using the Scatchard equation: r/c=K(n−r): where r=molesof bound ligand/mole of receptor at equilibrium; c=free ligandconcentration at equilibrium; K=equilibrium association constant; andn=number of ligand binding sites per receptor molecule. By graphicalanalysis, r/c is plotted on the Y-axis versus r on the X-axis, thusproducing a Scatchard plot. Antibody affinity measurement by Scatchardanalysis is well known in the art. See, e.g., van Erp et al., J.Immunoassay 12: 425-43, 1991; Nelson and Griswold, Comput. MethodsPrograms Biomed. 27: 65-8, 1988.

The term “epitope” refers to an antigenic determinant capable ofspecific binding to an antibody. Epitopes usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents.

Numerous publications discuss the use of phage display technology toproduce and screen libraries of polypeptides for binding to a selectedanalyte. See, e.g, Cwirla et al., Proc. Natl. Acad. Sci. USA 87,6378-82, 1990; Devlin et al., Science 249, 404-6, 1990, Scott and Smith,Science 249, 386-88, 1990; and Ladner et al., U.S. Pat. No. 5,571,698. Abasic concept of phage display methods is the establishment of aphysical association between DNA encoding a polypeptide to be screenedand the polypeptide. This physical association is provided by the phageparticle, which displays a polypeptide as part of a capsid enclosing thephage genome which encodes the polypeptide. The establishment of aphysical association between polypeptides and their genetic materialallows simultaneous mass screening of very large numbers of phagebearing different polypeptides. Phage displaying a polypeptide withaffinity to a target bind to the target and these phage are enriched byaffinity screening to the target. The identity of polypeptides displayedfrom these phage can be determined from their respective genomes. Usingthese methods a polypeptide identified as having a binding affinity fora desired target can then be synthesized in bulk by conventional means.See, e.g., U.S. Pat. No. 6,057,098, which is hereby incorporated in itsentirety, including all tables, figures, and claims.

The antibodies that are generated by these methods may then be selectedby first screening for affinity and specificity with the purifiedpolypeptide of interest and, if required, comparing the results to theaffinity and specificity of the antibodies with polypeptides that aredesired to be excluded from binding. The screening procedure can involveimmobilization of the purified polypeptides in separate wells ofmicrotiter plates. The solution containing a potential antibody orgroups of antibodies is then placed into the respective microtiter wellsand incubated for about 30 min to 2 h. The microtiter wells are thenwashed and a labeled secondary antibody (for example, an anti-mouseantibody conjugated to alkaline phosphatase if the raised antibodies aremouse antibodies) is added to the wells and incubated for about 30 minand then washed. Substrate is added to the wells and a color reactionwill appear where antibody to the immobilized polypeptide(s) arepresent.

The antibodies so identified may then be further analyzed for affinityand specificity in the assay design selected. In the development ofimmunoassays for a target protein, the purified target protein acts as astandard with which to judge the sensitivity and specificity of theimmunoassay using the antibodies that have been selected. Because thebinding affinity of various antibodies may differ; certain antibodypairs (e.g., in sandwich assays) may interfere with one anothersterically, etc., assay performance of an antibody may be a moreimportant measure than absolute affinity and specificity of an antibody.

While the present application describes antibody-based binding assays indetail, alternatives to antibodies as binding species in assays are wellknown in the art. These include receptors for a particular target,aptamers, etc. Aptamers are oligonucleic acid or peptide molecules thatbind to a specific target molecule. Aptamers are usually created byselecting them from a large random sequence pool, but natural aptamersalso exist. High-affinity aptamers containing modified nucleotidesconferring improved characteristics on the ligand, such as improved invivo stability or improved delivery characteristics. Examples of suchmodifications include chemical substitutions at the ribose and/orphosphate and/or base positions, and may include amino acid side chainfunctionalities. Assay Correlations

Assay Correlations

The term “correlating” as used herein in reference to the use ofbiomarkers refers to comparing the presence or amount of thebiomarker(s) in a patient to its presence or amount in persons known tosuffer from, or known to be at risk of, a given condition; or in personsknown to be free of a given condition. Often, this takes the form ofcomparing an assay result in the form of a biomarker concentration to apredetermined threshold selected to be indicative of the occurrence ornonoccurrence of a disease or the likelihood of some future outcome.

Selecting a diagnostic threshold involves, among other things,consideration of the probability of disease, distribution of true andfalse diagnoses at different test thresholds, and estimates of theconsequences of treatment (or a failure to treat) based on thediagnosis. For example, when considering administering a specifictherapy which is highly efficacious and has a low level of risk, fewtests are needed because clinicians can accept substantial diagnosticuncertainty. On the other hand, in situations where treatment optionsare less effective and more risky, clinicians often need a higher degreeof diagnostic certainty. Thus, cost/benefit analysis is involved inselecting a diagnostic threshold.

Suitable thresholds may be determined in a variety of ways. For example,one recommended diagnostic threshold for the diagnosis of acutemyocardial infarction using cardiac troponin is the 97.5^(th) percentileof the concentration seen in a normal population. Another method may beto look at serial samples from the same patient, where a prior“baseline” result is used to monitor for temporal changes in a biomarkerlevel.

Population studies may also be used to select a decision threshold.Reciever Operating Characteristic (“ROC”) arose from the field of signaldectection therory developed during World War II for the analysis ofradar images, and ROC analysis is often used to select a threshold ableto best distinguish a “diseased” subpopulation from a “nondiseased”subpopulation. A false positive in this case occurs when the persontests positive, but actually does not have the disease. A falsenegative, on the other hand, occurs when the person tests negative,suggesting they are healthy, when they actually do have the disease. Todraw a ROC curve, the true positive rate (TPR) and false positive rate(FPR) are determined as the decision threshold is varied continuously.Since TPR is equivalent with sensitivity and FPR is equal to1−specificity, the ROC graph is sometimes called the sensitivity vs(1−specificity) plot. A perfect test will have an area under the ROCcurve of 1.0; a random test will have an area of 0.5. A threshold isselected to provide an acceptable level of specificity and sensitivity.

In this context, “diseased” is meant to refer to a population having onecharacteristic (the presence of a disease or condition or the occurrenceof some outcome) and “nondiseased” is meant to refer to a populationlacking the characteristic. While a single decision threshold is thesimplest application of such a method, multiple decision thresholds maybe used. For example, below a first threshold, the absence of diseasemay be assigned with relatively high confidence, and above a secondthreshold the presence of disease may also be assigned with relativelyhigh confidence. Between the two thresholds may be consideredindeterminate. This is meant to be exemplary in nature only.

In addition to threshold comparisons, other methods for correlatingassay results to a patient classification (occurrence or nonoccurrenceof disease, likelihood of an outcome, etc.) include decision trees, rulesets, Bayesian methods, and neural network methods. These methods canproduce probability values representing the degree to which a subjectbelongs to one classification out of a plurality of classifications.

Measures of test accuracy may be obtained as described in Fischer etal., Intensive Care Med. 29: 1043-51, 2003, and used to determine theeffectiveness of a given biomarker. These measures include sensitivityand specificity, predictive values, likelihood ratios, diagnostic oddsratios, and ROC curve areas. The area under the curve (“AUC”) of a ROCplot is equal to the probability that a classifier will rank a randomlychosen positive instance higher than a randomly chosen negative one. Thearea under the ROC curve may be thought of as equivalent to theMann-Whitney U test, which tests for the median difference betweenscores obtained in the two groups considered if the groups are ofcontinuous data, or to the Wilcoxon test of ranks.

As discussed above, suitable tests may exhibit one or more of thefollowing results on these various measures: a specificity of greaterthan 0.5, preferably at least 0.6, more preferably at least 0.7, stillmore preferably at least 0.8, even more preferably at least 0.9 and mostpreferably at least 0.95, with a corresponding sensitivity greater than0.2, preferably greater than 0.3, more preferably greater than 0.4,still more preferably at least 0.5, even more preferably 0.6, yet morepreferably greater than 0.7, still more preferably greater than 0.8,more preferably greater than 0.9, and most preferably greater than 0.95;a sensitivity of greater than 0.5, preferably at least 0.6, morepreferably at least 0.7, still more preferably at least 0.8, even morepreferably at least 0.9 and most preferably at least 0.95, with acorresponding specificity greater than 0.2, preferably greater than 0.3,more preferably greater than 0.4, still more preferably at least 0.5,even more preferably 0.6, yet more preferably greater than 0.7, stillmore preferably greater than 0.8, more preferably greater than 0.9, andmost preferably greater than 0.95; at least 75% sensitivity, combinedwith at least 75% specificity; a ROC curve area of greater than 0.5,preferably at least 0.6, more preferably 0.7, still more preferably atleast 0.8, even more preferably at least 0.9, and most preferably atleast 0.95; an odds ratio different from 1, preferably at least about 2or more or about 0.5 or less, more preferably at least about 3 or moreor about 0.33 or less, still more preferably at least about 4 or more orabout 0.25 or less, even more preferably at least about 5 or more orabout 0.2 or less, and most preferably at least about 10 or more orabout 0.1 or less; a positive likelihood ratio (calculated assensitivity/(1−specificity)) of greater than 1, at least 2, morepreferably at least 3, still more preferably at least 5, and mostpreferably at least 10; and or a negative likelihood ratio (calculatedas (1−sensitivity)/specificity) of less than 1, less than or equal to0.5, more preferably less than or equal to 0.3, and most preferably lessthan or equal to 0.1

Additional clinical indicia may be combined with the kidney injurymarker assay result(s) of the present invention. These include otherbiomarkers related to renal status. Examples include the following,which recite the common biomarker name, followed by the Swiss-Prot entrynumber for that biomarker or its parent: Actin (P68133); Adenosinedeaminase binding protein (DPP4, P27487); Alpha-1-acid glycoprotein 1(P02763); Alpha-1-microglobulin (P02760); Albumin (P02768);Angiotensinogenase (Renin, P00797); Annexin A2 (P07355);Beta-glucuronidase (P08236); B-2-microglobulin (P61679);Beta-galactosidase (P16278); BMP-7 (P18075); Brain natriuretic peptide(proBNP, BNP-32, NTproBNP; P16860); Calcium-binding protein Beta(S100-beta, P04271); Carbonic anhydrase (Q16790); Casein Kinase 2(P68400); Cathepsin B (P07858); Ceruloplasmin (P00450); Clusterin(P10909); Complement C3 (P01024); Cysteine-rich protein (CYR61, O00622);Cytochrome C (P99999); Epidermal growth factor (EGF, P01133);Endothelin-1 (P05305); Exosomal Fetuin-A (P02765); Fatty acid-bindingprotein, heart (FABP3, P05413); Fatty acid-binding protein, liver(P07148); Ferritin (light chain, P02793; heavy chain P02794);Fructose-1,6-biphosphatase (P09467); GRO-alpha (CXCL1, (P09341); GrowthHormone (P01241); Hepatocyte growth factor (P14210); Insulin-like growthfactor I (P01343); Immunoglobulin G; Immunoglobulin Light Chains (Kappaand Lambda); Interferon gamma (P01308); Lysozyme (P61626);Interleukin-lalpha (P01583); Interleukin-2 (P60568); Interleukin-4(P60568); Interleukin-9 (P15248); Interleukin-12p40 (P29460);Interleukin-13 (P35225); Interleukin-16 (Q14005); L1 cell adhesionmolecule (P32004); Lactate dehydrogenase (P00338); LeucineAminopeptidase (P28838); Meprin A-alpha subunit (Q16819); Meprin A-betasubunit (Q16820); Midkine (P21741); MIP2-alpha (CXCL2, P19875); MMP-2(P08253); MMP-9 (P14780); Netrin-1 (O95631); Neutral endopeptidase(P08473); Osteopontin (P10451); Renal papillary antigen 1 (RPA1); Renalpapillary antigen 2 (RPA2); Retinol binding protein (P09455);Ribonuclease; S100 calcium-binding protein A6 (P06703); Serum Amyloid PComponent (P02743); Sodium/Hydrogen exchanger isoform (NHE3, P48764);Spermidine/spermine N1-acetyltransferase (P21673); TGF-Betal (P01137);Transferrin (P02787); Trefoil factor 3 (TFF3, Q07654); Toll-Like protein4 (O00206); Total protein; Tubulointerstitial nephritis antigen(Q9UJW2); Uromodulin (Tamm-Horsfall protein, P07911).

For purposes of risk stratification, Adiponectin (Q15848); Alkalinephosphatase (P05186); Aminopeptidase N (P15144); CalbindinD28k (P05937);Cystatin C (P01034); 8 subunit of F1FO ATPase (P03928);Gamma-glutamyltransferase (P19440); GSTa(alpha-glutathione-S-transferase, P08263); GSTpi(Glutathione-S-transferase P; GST class-pi; P09211); IGFBP-1 (P08833);IGFBP-2 (P18065); IGFBP-6 (P24592); Integral membrane protein 1 (Itm1,P46977); Interleukin-6 (P05231); Interleukin-8 (P10145); Interleukin-18(Q14116); IP-10 (10 kDa interferon-gamma-induced protein, P02778); IRPR(IFRD1, O00458); Isovaleryl-CoA dehydrogenase (IVD, P26440);I-TAC/CXCL11 (014625); Keratin 19 (P08727); Kim-1 (Hepatitis A viruscellular receptor 1, O43656); L-arginine:glycine amidinotransferase(P50440); Leptin (P41159); Lipocalin2 (NGAL, P80188); MCP-1 (P13500);MIG (Gamma-interferon-induced monokine Q07325); MIP-1a (P10147); MIP-3a(P78556); MIP-lbeta (P13236); MIP-1d (Q16663); NAG(N-acetyl-beta-D-glucosaminidase, P54802); Organic ion transporter(OCT2, O15244); Osteoprotegerin (O14788); P8 protein (O60356);Plasminogen activator inhibitor 1 (PAI-1, P05121); ProANP(1-98)(P01160); Protein phosphatase 1-beta (PPI-beta, P62140); Rab GDI-beta(P50395); Renal kallikrein (Q86U61); RT1.B-1 (alpha) chain of theintegral membrane protein (Q5Y7A8); Soluble tumor necrosis factorreceptor superfamily member 1A (sTNFR-I, P19438); Soluble tumor necrosisfactor receptor superfamily member 1B (sTNFR-II, P20333); Tissueinhibitor of metalloproteinases 3 (TIMP-3, P35625); uPAR (Q03405) may becombined with the kidney injury marker assay result(s) of the presentinvention.

Other clinical indicia which may be combined with the kidney injurymarker assay result(s) of the present invention includes demographicinformation (e.g., weight, sex, age, race), medical history (e.g.,family history, type of surgery, pre-existing disease such as aneurism,congestive heart failure, preeclampsia, eclampsia, diabetes mellitus,hypertension, coronary artery disease, proteinuria, renal insufficiency,or sepsis, type of toxin exposure such as NSAIDs, cyclosporines,tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin,myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrastagents, or streptozotocin), clinical variables (e.g., blood pressure,temperature, respiration rate), risk scores (APACHE score, PREDICTscore, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), a urinetotal protein measurement, a glomerular filtration rate, an estimatedglomerular filtration rate, a urine production rate, a serum or plasmacreatinine concentration, a renal papillary antigen 1 (RPA1)measurement; a renal papillary antigen 2 (RPA2) measurement; a urinecreatinine concentration, a fractional excretion of sodium, a urinesodium concentration, a urine creatinine to serum or plasma creatinineratio, a urine specific gravity, a urine osmolality, a urine ureanitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnine ratio,and/or a renal failure index calculated as urine sodium/(urinecreatinine/plasma creatinine). Other measures of renal function whichmay be combined with the kidney injury marker assay result(s) aredescribed hereinafter and in Harrison's Principles of Internal Medicine,17^(th) Ed., McGraw Hill, N.Y., pages 1741-1830, and Current MedicalDiagnosis & Treatment 2008, 47^(th) Ed, McGraw Hill, N.Y., pages785-815, each of which are hereby incorporated by reference in theirentirety.

Combining assay results/clinical indicia in this manner can comprise theuse of multivariate logistical regression, loglinear modeling, neuralnetwork analysis, n-of-m analysis, decision tree analysis, etc. Thislist is not meant to be limiting.

Diagnosis of Acute Renal Failure

As noted above, the terms “acute renal (or kidney) injury” and “acuterenal (or kidney) failure” as used herein are defined in part in termsof changes in serum creatinine from a baseline value. Most definitionsof ARF have common elements, including the use of serum creatinine and,often, urine output. Patients may present with renal dysfunction withoutan available baseline measure of renal function for use in thiscomparison. In such an event, one may estimate a baseline serumcreatinine value by assuming the patient initially had a normal GFR.Glomerular filtration rate (GFR) is the volume of fluid filtered fromthe renal (kidney) glomerular capillaries into the Bowman's capsule perunit time. Glomerular filtration rate (GFR) can be calculated bymeasuring any chemical that has a steady level in the blood, and isfreely filtered but neither reabsorbed nor secreted by the kidneys. GFRis typically expressed in units of ml/min:

${G\; F\; R} = \frac{{Urine}\mspace{14mu} {Concentration} \times {Urine}\mspace{14mu} {Flow}}{{Plasma}\mspace{14mu} {Concentration}}$

By normalizing the GFR to the body surface area, a GFR of approximately75-100 ml/min per 1.73 m² can be assumed. The rate therefore measured isthe quantity of the substance in the urine that originated from acalculable volume of blood.

There are several different techniques used to calculate or estimate theglomerular filtration rate (GFR or eGFR). In clinical practice, however,creatinine clearance is used to measure GFR. Creatinine is producednaturally by the body (creatinine is a metabolite of creatine, which isfound in muscle). It is freely filtered by the glomerulus, but alsoactively secreted by the renal tubules in very small amounts such thatcreatinine clearance overestimates actual GFR by 10-20%. This margin oferror is acceptable considering the ease with which creatinine clearanceis measured.

Creatinine clearance (CCr) can be calculated if values for creatinine'surine concentration (U_(Cr)), urine flow rate (V), and creatinine'splasma concentration (P_(Cr)) are known. Since the product of urineconcentration and urine flow rate yields creatinine's excretion rate,creatinine clearance is also said to be its excretion rate (U_(Cr)×V)divided by its plasma concentration. This is commonly representedmathematically as:

$C_{Cr} = \frac{U_{Cr} \times V}{P_{Cr}}$

Commonly a 24 hour urine collection is undertaken, from empty-bladderone morning to the contents of the bladder the following morning, with acomparative blood test then taken:

$C_{Cr} = \frac{U_{Cr} \times 24\text{-}{hour}\mspace{14mu} {volume}}{P_{Cr} \times 24 \times 60\mspace{14mu} {mins}}$

To allow comparison of results between people of different sizes, theCCr is often corrected for the body surface area (BSA) and expressedcompared to the average sized man as ml/min/1.73 m2. While most adultshave a BSA that approaches 1.7 (1.6-1.9), extremely obese or slimpatients should have their CCr corrected for their actual BSA:

$C_{{Cr} - {corrected}} = \frac{C_{Cr} \times 1.73}{BSA}$

The accuracy of a creatinine clearance measurement (even when collectionis complete) is limited because as glomerular filtration rate (GFR)falls creatinine secretion is increased, and thus the rise in serumcreatinine is less. Thus, creatinine excretion is much greater than thefiltered load, resulting in a potentially large overestimation of theGFR (as much as a twofold difference). However, for clinical purposes itis important to determine whether renal function is stable or gettingworse or better. This is often determined by monitoring serum creatininealone. Like creatinine clearance, the serum creatinine will not be anaccurate reflection of GFR in the non-steady-state condition of ARF.Nonetheless, the degree to which serum creatinine changes from baselinewill reflect the change in GFR. Serum creatinine is readily and easilymeasured and it is specific for renal function.

For purposes of determining urine output on a Urine output on a mL/kg/hrbasis, hourly urine collection and measurement is adequate. In the casewhere, for example, only a cumulative 24-h output was available and nopatient weights are provided, minor modifications of the RIFLE urineoutput criteria have been described. For example, Bagshaw et al.,Nephrol. Dial. Transplant. 23: 1203-1210, 2008, assumes an averagepatient weight of 70 kg, and patients are assigned a RIFLEclassification based on the following: <35 mL/h (Risk), <21 mL/h(Injury) or <4 mL/h (Failure).

Selecting a Treatment Regimen

Once a diagnosis is obtained, the clinician can readily select atreatment regimen that is compatible with the diagnosis, such asinitiating renal replacement therapy, withdrawing delivery of compoundsthat are known to be damaging to the kidney, kidney transplantation,delaying or avoiding procedures that are known to be damaging to thekidney, modifying diuretic administration, initiating goal directedtherapy, etc. The skilled artisan is aware of appropriate treatments fornumerous diseases discussed in relation to the methods of diagnosisdescribed herein. See, e.g., Merck Manual of Diagnosis and Therapy, 17thEd. Merck Research Laboratories, Whitehouse Station, N.J., 1999. Inaddition, since the methods and compositions described herein provideprognostic information, the markers of the present invention may be usedto monitor a course of treatment. For example, improved or worsenedprognostic state may indicate that a particular treatment is or is notefficacious.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The examples providedherein are representative of preferred embodiments, are exemplary, andare not intended as limitations on the scope of the invention.

EXAMPLE 1 Contrast-Induced Nephropathy Sample Collection

The objective of this sample collection study is to collect samples ofplasma and urine and clinical data from patients before and afterreceiving intravascular contrast media. Approximately 250 adultsundergoing radiographic/angiographic procedures involving intravascularadministration of iodinated contrast media are enrolled. To be enrolledin the study, each patient must meet all of the following inclusioncriteria and none of the following exclusion criteria:

Inclusion Criteria

-   males and females 18 years of age or older;-   undergoing a radiographic/angiographic procedure (such as a CT scan    or coronary intervention) involving the intravascular administration    of contrast media;-   expected to be hospitalized for at least 48 hours after contrast    administration.-   able and willing to provide written informed consent for study    participation and to comply with all study procedures.

Exclusion Criteria

-   renal transplant recipients;-   acutely worsening renal function prior to the contrast procedure;-   already receiving dialysis (either acute or chronic) or in imminent    need of dialysis at enrollment;-   expected to undergo a major surgical procedure (such as involving    cardiopulmonary bypass) or an additional imaging procedure with    contrast media with significant risk for further renal insult within    the 48 hrs following contrast administration;-   participation in an interventional clinical study with an    experimental therapy within the previous 30 days;-   known infection with human immunodeficiency virus (HIV) or a    hepatitis virus.

Immediately prior to the first contrast administration (and after anypre-procedure hydration), an EDTA anti-coagulated blood sample (10 mL)and a urine sample (10 mL) are collected from each patient. Blood andurine samples are then collected at 4 (±0.5), 8 (±1), 24 (±2) 48 (±2),and 72 (±2) hrs following the last administration of contrast mediaduring the index contrast procedure. Blood is collected via directvenipuncture or via other available venous access, such as an existingfemoral sheath, central venous line, peripheral intravenous line orhep-lock. These study blood samples are processed to plasma at theclinical site, frozen and shipped to Astute Medical, Inc., San Diego,Calif. The study urine samples are frozen and shipped to Astute Medical,Inc.

Serum creatinine is assessed at the site immediately prior to the firstcontrast administration (after any pre-procedure hydration) and at 4(±0.5), 8 (±1), 24 (±2) and 48 (±2)), and 72 (±2) hours following thelast administration of contrast (ideally at the same time as the studysamples are obtained). In addition, each patient's status is evaluatedthrough day 30 with regard to additional serum and urine creatininemeasurements, a need for dialysis, hospitalization status, and adverseclinical outcomes (including mortality).

Prior to contrast administration, each patient is assigned a risk basedon the following assessment: systolic blood pressure <80 mm Hg=5 points;intra-arterial balloon pump=5 points; congestive heart failure (ClassIII-IV or history of pulmonary edema)=5 points; age >75 yrs=4 points;hematocrit level <39% for men, <35% for women=3 points; diabetes=3points; contrast media volume=1 point for each 100 mL; serum creatininelevel >1.5 g/dL=4 points OR estimated GFR 40-60 mL/min/1.73 m²=2 points,20-40 mL/min/1.73 m²=4 points, <20 mL/min/1.73 m²=6 points. The risksassigned are as follows: risk for CIN and dialysis: 5 or less totalpoints=risk of CIN −7.5%, risk of dialysis −0.04%; 6-10 totalpoints=risk of CIN −14%, risk of dialysis −0.12%; 11-16 totalpoints=risk of CIN −26.1%, risk of dialysis −1.09%; >16 totalpoints=risk of CIN −57.3%, risk of dialysis −12.8%.

EXAMPLE 2 Cardiac Surgery Sample Collection

The objective of this sample collection study is to collect samples ofplasma and urine and clinical data from patients before and afterundergoing cardiovascular surgery, a procedure known to be potentiallydamaging to kidney function. Approximately 900 adults undergoing suchsurgery are enrolled. To be enrolled in the study, each patient mustmeet all of the following inclusion criteria and none of the followingexclusion criteria:

Inclusion Criteria

-   males and females 18 years of age or older;-   undergoing cardiovascular surgery;-   Toronto/Ottawa Predictive Risk Index for Renal Replacement risk    score of at least 2 (Wijeysundera et al., JAMA 297: 1801-9, 2007);    and-   able and willing to provide written informed consent for study    participation and to comply with all study procedures.

Exclusion Criteria

-   known pregnancy;-   previous renal transplantation;-   acutely worsening renal function prior to enrollment (e.g., any    category of RIFLE criteria);-   already receiving dialysis (either acute or chronic) or in imminent    need of dialysis at enrollment;-   currently enrolled in another clinical study or expected to be    enrolled in another clinical study within 7 days of cardiac surgery    that involves drug infusion or a therapeutic intervention for AKI;-   known infection with human immunodeficiency virus (HIV) or a    hepatitis virus.

Within 3 hours prior to the first incision (and after any pre-procedurehydration), an EDTA anti-coagulated blood sample (10 mL), whole blood (3mL), and a urine sample (35 mL) are collected from each patient. Bloodand urine samples are then collected at 3 (±0.5), 6 (±0.5), 12 (±1), 24(±2) and 48 (±2) hrs following the procedure and then daily on days 3through 7 if the subject remains in the hospital. Blood is collected viadirect venipuncture or via other available venous access, such as anexisting femoral sheath, central venous line, peripheral intravenousline or hep-lock. These study blood samples are frozen and shipped toAstute Medical, Inc., San Diego, Calif. The study urine samples arefrozen and shipped to Astute Medical, Inc.

EXAMPLE 3 Acutely Ill Subject Sample Collection

The objective of this study is to collect samples from acutely illpatients. Approximately 900 adults expected to be in the ICU for atleast 48 hours will be enrolled. To be enrolled in the study, eachpatient must meet all of the following inclusion criteria and none ofthe following exclusion criteria:

Inclusion Criteria

-   males and females 18 years of age or older;-   Study population 1: approximately 300 patients that have at least    one of:-   shock (SBP <90 mmHg and/or need for vasopressor support to maintain    MAP >60 mmHg and/or documented drop in SBP of at least 40 mmHg); and-   sepsis;-   Study population 2: approximately 300 patients that have at least    one of:-   IV antibiotics ordered in computerized physician order entry (CPOE)    within 24 hours of enrollment;-   contrast media exposure within 24 hours of enrollment;-   increased Intra-Abdominal Pressure with acute decompensated heart    failure; and-   severe trauma as the primary reason for ICU admission and likely to    be hospitalized in the ICU for 48 hours after enrollment;-   Study population 3: approximately 300 patients-   expected to be hospitalized through acute care setting (ICU or ED)    with a known risk factor for acute renal injury (e.g. sepsis,    hypotension/shock (Shock=systolic BP<90 mmHg and/or the need for    vasopressor support to maintain a MAP >60 mmHg and/or a documented    drop in SBP >40 mmHg), major trauma, hemorrhage, or major surgery);    and/or expected to be hospitalized to the ICU for at least 24 hours    after enrollment.

Exclusion Criteria

-   known pregnancy;-   institutionalized individuals;-   previous renal transplantation;-   known acutely worsening renal function prior to enrollment (e.g.,    any category of RIFLE criteria);-   received dialysis (either acute or chronic) within 5 days prior to    enrollment or in imminent need of dialysis at the time of    enrollment;-   known infection with human immunodeficiency virus (HIV) or a    hepatitis virus;-   meets only the SBP <90 mmHg inclusion criterion set forth above, and    does not have shock in the attending physician's or principal    investigator's opinion.

After providing informed consent, an EDTA anti-coagulated blood sample(10 mL) and a urine sample (25-30 mL) are collected from each patient.Blood and urine samples are then collected at 4 (±0.5) and 8 (±1) hoursafter contrast administration (if applicable); at 12 (±1), 24 (±2), and48 (±2) hours after enrollment, and thereafter daily up to day 7 to day14 while the subject is hospitalized. Blood is collected via directvenipuncture or via other available venous access, such as an existingfemoral sheath, central venous line, peripheral intravenous line orhep-lock. These study blood samples are processed to plasma at theclinical site, frozen and shipped to Astute Medical, Inc., San Diego,Calif. The study urine samples are frozen and shipped to Astute Medical,Inc.

EXAMPLE 4 Immunoassay Format

Analytes are is measured using standard sandwich enzyme immunoassaytechniques. A first antibody which binds the analyte is immobilized inwells of a 96 well polystyrene microplate. Analyte standards and testsamples are pipetted into the appropriate wells and any analyte presentis bound by the immobilized antibody. After washing away any unboundsubstances, a horseradish peroxidase-conjugated second antibody whichbinds the analyte is added to the wells, thereby forming sandwichcomplexes with the analyte (if present) and the first antibody.Following a wash to remove any unbound antibody-enzyme reagent, asubstrate solution comprising tetramethylbenzidine and hydrogen peroxideis added to the wells. Color develops in proportion to the amount ofanalyte present in the sample. The color development is stopped and theintensity of the color is measured at 540 nm or 570 nm. An analyteconcentration is assigned to the test sample by comparison to a standardcurve determined from the analyte standards.

EXAMPLE 5 Apparently Healthy Donor and Chronic Disease Patient Samples

Human urine samples from donors with no known chronic or acute disease(“Apparently Healthy Donors”) were purchased from two vendors (GoldenWest Biologicals, Inc., 27625 Commerce Center Dr., Temecula, Calif.92590 and Virginia Medical Research, Inc., 915 First Colonial Rd.,Virginia Beach, Va. 23454). The urine samples were shipped and storedfrozen at less than −20° C. The vendors supplied demographic informationfor the individual donors including gender, race (Black/White), smokingstatus and age.

Human urine samples from donors with various chronic diseases (“ChronicDisease Patients”) including congestive heart failure, coronary arterydisease, chronic kidney disease, chronic obstructive pulmonary disease,diabetes mellitus and hypertension were purchased from Virginia MedicalResearch, Inc., 915 First Colonial Rd., Virginia Beach, Va. 23454. Theurine samples were shipped and stored frozen at less than -20 degreescentigrade. The vendor provided a case report form for each individualdonor with age, gender, race (Black/White), smoking status and alcoholuse, height, weight, chronic disease(s) diagnosis, current medicationsand previous surgeries.

EXAMPLE 6 Biomarker Panels

The following exemplary method was used to segregate a patientpopulation into two groups, which will be referred to for convenience as“non-diseased” (NonDis) and “diseased” (Dis). The combined NonDis/Dispopulation formed a data set, and the following analysis was performed.Multiple analytes were measured from either blood or urine or both, forthe subjects in the data set. Blood and urine measurements were treatedas separate biomarkers in calculating panel results. Panels were formedby algorithmically combing two or more analytes in one of two ways: (i)a very simple product and division of analyte values; and (ii) logisticregression.

Logistic regression is a method widely used for models which have abinary outcome, such as the “diseased” “non-diseased” dichotomypresented here. The method will be briefly described below, fulltreatments can be found in the literature. The model used is:

${\Pi_{i}\left( {y_{i} = \left. 1 \middle| x_{i} \right.} \right)} = \frac{^{\alpha + {\beta \; x_{i}}}}{1 + {^{\alpha + \beta}\; x_{i}}}$

In this model, x and β are vectors, x representing the differentobservables or analyte values, y_(i)=1 indicates a diseased state, andπ_(i) is the model probability of this state for the i^(th) case givenx_(i). The panel value for each sample is π_(i). The log odds or logitis:

${logit} = {{\ln \left\lbrack \frac{\Pi_{i}\left( {y_{i} = \left. 1 \middle| x_{i} \right.} \right)}{1 - {\Pi_{i}\left( {y_{i} = \left. 1 \middle| x_{i} \right.} \right)}} \right\rbrack} = {\alpha + {\beta \; x_{i}}}}$

Define p_(i) as probability of observing the true outcome:

$p_{i} = \begin{Bmatrix}{\Pi_{i}\left( {y_{i} = \left. 1 \middle| x_{i} \right.} \right)} & {{if}\mspace{14mu} {deseased}} \\{1 - {\Pi_{i}\left( {y_{i} = \left. 1 \middle| x_{i} \right.} \right)}} & {{if}\mspace{14mu} {non}\text{-}{diseased}}\end{Bmatrix}$

The likelihood function is the product of the probabilities of observingthe true outcomes, so the log likelihood (LL) is:

LL=ln(L(α,β)=ρln(p _(i))

To find the parameters, α and β, that best fit the model, the negativelog likelihood (-LL) is minimized. This minimization was performed usingthe Levenberg-Marquardt method (Numerical Recipes The Art of ScientificComputing, Third Edition, Cambridge University Press, 2007). The initialpoint for each parameter is 0.

A commonly used statistic to compare the fit of two nested models is thelikelihood ratio test. This statistic, or the deviance, is thedifference in twice the negative log likelihood (−2LL) for the twomodel, and is asymptotically a χ² with DF equal to the change in thenumber of degrees of freedom (number of analytes) between the models.The p-value is calculated from this statistic. The null hypothesis isthat the logistic model is not different than a constant model (β set to0), is tested for each model using the likelihood ratio test. The ‘modelp-value’ is defined as the probability that the null hypothesis is true.For the constant model, a closed form solution for α and −2LL can befound. It is a function of the number of diseased (#D) and the number ofnon-diseased (#ND) samples in the data set.

$\alpha = {{{\ln \left( \frac{\# \; D}{\# \; {ND}} \right)} - {2\; {LL}}} = {{- 2}*\left\lbrack {{\# \; {ND}\; {\ln \left( {1 - \Pi} \right)}} + {\# \; D\; {\ln (\Pi)}}} \right\rbrack}}$Where:$\Pi = \frac{\# \; D}{\left( {{\# \; {ND}} + {\# \; D}} \right)}$

Each analyte can be tested for significance by holding its β at zero andcomparing the model found without the analyte to the full model. Thelikelihood ratio test is used with 1 degree of freedom. The ‘analytep-value’ is defined as the probability that the models are not differentwhen that analyte is removed.

The analyte concentrations used in the panel can either be logtransformed or untransformed. For each analyte in the panel, all thepermutations of the analytes being log transformed/not transformed arecalculated. The permutation with the lowest model p-value is used forthat panel. For two marker panels there are 4 permutations, for sixmarker panels there are 8.

Table 3 contains a list of assays that were measured on urine (“U”) andEDTA plasmas (“E”) samples. Also listed, are the two letter codes thatwill be used throughout this document to refer to these assays accordingto the following convention: if the first character of the code iscapitalized, this means the assay was measured in urine. If the secondcharacter of the code is capitalized, this means the assay was measuredin EDTA plasma. If both characters of the code are capitalized, then theassay was measured in both urine and EDTA plasma. In the table someassays have a single code in capitals, indicating that the same assayformat (e.g., microtiter dish, automated analyzer, etc.) was used forboth urine and plasma. Other assays have separate U and E codes. Thisindicates that different assay formats were used for urine and EDTAplasma. A code of “**” indicates that an insufficient number of sampleswere measured on that assay and it will not appear in the panel resultspresented hereinafter.

TABLE 3 Swiss- Code Swiss- Code Preferred Name Prot: U, E Preferred NameProt: U, E 60 kDa heat shock protein, P10809 YH 72 kDa type IVcollagenase P08253 CP mitochondrial 72 kDa type IV P08253 FE 72 kDa typeIV P08253 FD collagenase: Metalloproteinase P01033 collagenase:Metalloproteinase P16035 inhibitor 1 complex inhibitor 2 complex 72 kDatype IV P08253 FF Adiponectin Q15848 AD collagenase: MetalloproteinaseQ99727 inhibitor 4 complex Advanced glycosylation end Q15109 KKAgouti-related protein 000253 SI product-specific receptor Alkalinephosphatase, tissue- P05186 UL Alpha-l-antitrypsin P01009 Jj, aCnonspecific isozyme Alpha-l-antitrypsin Neutrophil P01009 WNAlpha-l-antitrypsin Plasminogen P01009 TK elastase complex P08246complex P00747 Alpha-2 macroglobulin P01023 Ir, aEAlpha-2-HS-glycoprotein P02765 IO Alpha-fetoprotein P02771 AFAmphiregulin P15514 TN Amyloid Beta 40 P05067 YE Amyloid Beta 42 P05067TM (aa672- (aa672- 711) 713) Angiogenin P03950 Tz, w Angiopoietin-1Q15389 DR J Angiopoietin-1 receptor Q02763 Us, gV Angiopoietin-2 015123HW Angiopoietin-related protein 3 Q9Y5C1 WC Angiopoietin-related protein4 Q9BY76 WD Angiopoietin-related protein 6 Q8NI99 WE Anti-Cathepsin-G(ANCA) NA DS Antileukoproteinase P03973 GL Apolipoprotein A-I P02647 Il,aG Apolipoprotein A-II P02652 IJ Apolipoprotein B-100 P04114 IHApolipoprotein C-III P02656 Ik, aH Apolipoprotein E P02649 In, qIApolipoprotein(a) P08519 It, cJ Appetite-regulating hormone Q9UBU3 RAAspartate aminotransferase, P17174 DB Bactericidal permeability- P17213DU cytoplasmic increasing protein Bc12 antagonist of cell death Q92934DQ Beta-2-glycoprotein 1 P02749 Im, aI Beta-2-microglobulin P61769 AJBeta-nerve growth factor P01138 Vz, pY Betacellulin P35070 TO Bonemorphogenetic protein 7 P18075 UO Brain-derived neurotrophic factorP23560 Jn, aK C-C motif chemokine 1 P22362 MU C-C motif chemokine 13Q99616 Mq, oT C-C motif chemokine 15 Q16663 MW C-C motif chemokine 17Q92583 Mx, oV C-C motif chemokine 18 P55774 JL C-C motif chemokine 19Q99731 Nq, iA C-C motif chemokine 2 P13500 Ou, cL C-C motif chemokine 20P78556 NR C-C motif chemokine 21 000585 MY C-C motif chemokine 22 O00626Mj, cM C-C motif chemokine 23 P55773 VI C-C motif chemokine 24 O00175 MZC-C motif chemokine 26 Q9Y258 NA C-C motif chemokine 27 Q9Y4X3 NB C-Cmotif chemokine 3 P10147 Mk, c N C-C motif chemokine 4 P13236 CO C-Cmotif chemokine 5 P13501 Js, cY C-C motif chemokine 7 P80098 MI C-Cmotif chemokine 8 P80075 MP C-Peptide P01308( QV C-reactive proteinP02741 IP aa57- 87) C-X-C motif chemokine 10 P02778 OW C-X-C motifchemokine 11 O14625 OH C-X-C motif chemokine 13 043927 MT C-X-C motifchemokine 16 Q9H2A7 EF C-X-C motif chemokine 2 P19875 PO C-X-C motifchemokine 5 P42830 Mr, aV C-X-C motif chemokine 6 P80162 NO C-X-C motifchemokine 9 Q07325 NN Cadherin-1 P12830 Gd, oO Cadherin-16 O75309 THCadherin-3 P22223 UG Cadherin-5 P33151 GT Calbindin P05937 YD CalcitoninP01258 AO Calcitonin (Procalcitonin) P01258- HO Cancer Antigen 15-3 NAIC Pro Cancer Antigen 19-9 NA AN Carbonic anhydrase 9 Q16790 VHCarcinoembryonic antigen-related P13688 VT Carcinoembryonicantigen-related P06731 AR cell adhesion molecule 1 cell adhesionmolecule 5 Caspase- 1 P29466 DV Caspase-3, active P42574 Kx, dXCaspase-8 Q14790 DW Caspase-9 P55211 DO Cathepsin B P07858 UT CathepsinD P07339 JP Cathepsin S P25774 UW CD40 ligand P29965 Ml, aQ CD44 antigenP16070 GC Cellular tumor antigen p53 P04637 RX Choriogonadotropinsubunit beta P01233 Zx, zH Ciliary neurotrophic factor P26441 SJClusterin P10909 Ii, sO Coagulation factor VII P08709 BB Collagenase 3P45452 Lt,1O Complement C3 P01024 AL Complement C4-B P0C0L5 JMComplement C5 P01031 VU Complement factor H P08603 QG CorticotropinP01189(JY aa138- 176) Cortisol NA Rh, rP Creatine Kinase-MB P12277 ASP06732 Creatinine NA AA Cyclin-dependent kinase inhibitor P38936 Fw, rW1 Cystatin-C P01034 IZ Cytochrome c P99999 KY DDRGK domain-containingQ96HY6 WM Dipeptidyl peptidase 4 P27487 VQ protein 1 E-selectin P16581PH Endoglin P17813 UP Endostatin P39060( Ua,wK Endothelial protein Creceptor Q9UNN8 Vj, eC aa1572- 1754) Endothelin-1 P05305 AW EotaxinP51671 Or, aY Epidermal growth factor receptor P00533 Kd, tQ EpiregulinO14944 TR Epithelial cell adhesion molecule P16422 UV ErythropoietinP01588 Uk, aZ Erythropoietin receptor P19235 ED Fatty acid-bindingprotein, heart P05413 BA Fatty acid-binding protein, P12104 EQ Fattyacid-binding protein, liver P07148 EZ intestinal Ferritin P02792 BCFibrinogen P02671 Iq, bE P02794 P02675 P02679 Fibroblast growth factor19 O95750 WG Fibroblast growth factor 21 Q9NSA1 WH Fibroblast growthfactor 23 Q9GZV9 WF Fibronectin P02751 Rm, jB Follistatin P19883 HXFollitropin P01215 JU P01225 Follitropin subunit beta P01225 SFFractalkine P78423 UM Galectin-3 P17931 VB Gastric inhibitorypolypeptide P09681 QY Glial cell line-derived P39905 TL Glial fibrillaryacidic protein P14136 EH neurotrophic factor Glucagon P01275 QZGlucagon-like peptide 1 P01275( QX aa98- 127 aa98- 128) GlutathioneS-transferase A1 P08263 Dp, bI Glutathione S-transferase P P09211 FYGranulocyte colony-stimulating P09919 Hq, bF Granulocyte-macrophagecolony- P04141 Pd, bH factor stimulating factor Granzyme B P10144 EOGranzyme M P51124 YF Growth-regulated alpha protein P09341 Tj, uIHaptoglobin P00738 Iu, bJ Heat shock 70 kDa protein 1 P08107 Yi, eP Heatshock protein beta-1 P04792 Yg, rS Heat shock protein beta-1 P04792 YKHeat shock protein HSP 90-alpha P07900 YJ (phospho SER78 / phospho(pS78/p SER82) S82) Heme oxygenase 1 P09601 EM Heparan Sulfate NA TIHeparin-binding EGF-like growth Q99075 TT Heparin-binding growth factor1 P05230 Ub, wL factor Heparin-binding growth factor 2 P09038 Lv, tSHepatitis A virus cellular receptor 043656 Uh, tF 1 Hepatocyte growthfactor P14210 OK Hepatocyte growth factor receptor P08581 RT Hyaluronicacid NA ET Hypoxia-inducible factor 1 alpha Q16665 OE Immunoglobulin ANA Qe, bM Immunoglobulin E NA BN Immunoglobulin M NA Pz, bPImmunoglogulin G1 NA QA Immunoglogulin G2 NA QD Immunoglogulin G3 NA QBImmunoglogulin G4 NA QC Insulin P01308 CH Insulin receptor substrate 1P35568 RU Insulin-like growth factor 1 P08069 RY receptor Insulin-likegrowth factor IA P01343 BO Insulin-like growth factor-binding P08833 OFprotein 1 Insulin-like growth factor-binding P18065 JD Insulin-likegrowth factor-binding P17936 JG protein 2 protein 3 Insulin-like growthfactor-binding P22692 JE Insulin-like growth factor-binding P24593 JFprotein 4 protein 5 Insulin-like growth factor-binding P24592 JHInsulin-like growth factor-binding Q16270 JI protein 6 protein 7Intercellular adhesion molecule 1 P05362 JO Intercellular adhesionmolecule 2 P13598 GN Intercellular adhesion molecule 3 P32942 PKInterferon alpha-2 P01563 LY Interferon gamma P01579 Pc, bLInterleukin-1 alpha P01583 Lz, bX Interleukin-1 beta P01584 PFInterleukin-1 receptor antagonist P18510 Ma, bZ protein Interleukin-1receptor type I P14778 KF Interleukin-1 receptor type II P27930 KGInterleukin-10 P22301 Pb, bQ Interleukin-11 P20809 NT Interleukin-12P29459 Oz, bS Interleukin-12 subunit beta P29460 Oi, bR P29460Interleukin-13 P35225 MF Interleukin-15 P40933 Mg, bU Interleukin-17AQ16552 MH Interleukin-18 Q14116 Uf, bW Interleukin-2 P60568 Pa, cAInterleukin-2 receptor alpha chain P01589 MM Interleukin-20 Q9NYY1 NIInterleukin-21 Q9HBE4 NJ Interleukin-23 Q9NPF7 NC Interleukin-28A Q8IZJONK P29460 Interleukin-29 Q81U54 NU Interleukin-3 P08700 Mb, cBInterleukin-33 095760 NL Interleukin-4 P05112 Mc, cC Interleukin-4receptor alpha chain P24394 KI Interleukin-5 P05113 Og, cD Interleukin-6P05231 Pg, cE Interleukin-6 receptor subunit P08887 Kj, oD alphaInterleukin-6 receptor subunit beta P40189 KE Interleukin-7 P13232 Md,cF Interleukin-8 P10145 Pe, cG Interleukin-9 P15248 ME Interstitialcollagenase P03956 Lp,lK Interstitial P03956 FC collagenase:Metalloproteinase P16035 inhibitor 2 complex Involucrin P07476 Rf, rNIslet amyloid polypeptide P10997 QW Keratin, type I cytoskeletal 19P08727 Zw, zG Keratin, type II cytoskeletal 1; P04264 Rj, rR (aa311-367)typel cytoskeletal 10 (Keratin-1, P13645 -10 mix) Keratin, type IIcytoskeletal 6 (6A, P02538 Ri, rQ Kit ligand P21583 Ng, dA -6B, -6C mix)P04259 P48668 Lactotransferrin P02788 FA Leptin P41159 Hu, cI Leukemiainhibitory factor P15018 ND Lipopolysaccharide (serotypes- NA Rg, rOK,-O) Lutropin P01215 JV Lutropin subunit beta P01229 SH P01229Lymphatic vessel endothelial Q9Y5Y7 VW Lymphotactin P47992 Ns, cKhyaluronic acid receptor 1 Lymphotoxin-alpha P01374 DI Lysozyme C P61626FB Macrophage colony-stimulating P09603 NM Macrophage metalloelastaseP39900 Ld, lN factor 1 Macrophage migration inhibitory P14174 KSMalondialdehyde-modified low- NA HF factor density lipoproteinMatrilysin P09237 Lh, lL Matrix metalloproteinase-9 P14780 Lj, cR Matrixmetalloproteinase- P14780 Oa, ** Matrix metalloproteinase- P14780 OB 9:Metalloproteinase inhibitor 2 P16035 9: Metalloproteinase inhibitor 3P35625 complex complex Metalloproteinase inhibitor 1 P01033 Nv, dFMetalloproteinase inhibitor 2 P16035 NW Metalloproteinase inhibitor 3P35625 Nx, sC Metalloproteinase inhibitor 4 Q99727 NY Midkine P21741 EXMix of Growth-regulated alpha, P09341 LX beta, and gamma proteins P19875P19876 Monocyte differentiation antigen P08571 VO Mucin-16 Q8WXI7 Id, aMCD14 Myeloid differentiation primary Q99836 VC Myeloperoxidase P05164 CSresponse protein MyD88 Myoglobin P02144 CT Neprilysin P08473 UY Netrin-1O95631 FN Neural cell adhesion molecule 1 P13591 JT Neuronal celladhesion molecule Q92823 UZ Neutrophil collagenase P22894 Li, lMNeutrophil elastase P08246 FP Neutrophil gelatinase-associated P80188 FRlipocalin NF-kappa-B inhibitor alpha P25963 RV Nidogen-1 P14543 VSNitric oxide synthase, inducible P35228 EW NT-pro-BNP P16860 HCOsteocalcin P02818 RC Osteopontin P10451 OP Oxidized low-densitylipoprotein P78380 HB P-selectin P16109 PI receptor 1 P-selectinglycoprotein ligand 1 Q14242 GP Pancreatic prohormone P01298 QUPappalysin-1 Q13219 CW Parathyroid hormone P01270 RB Peptide YY P10082QT Pigment epithelium-derived factor P36955 JK Placenta growth factorP49763 Ue, tU Plasminogen activator inhibitor 1 P05121 CU Platelet basicprotein P02775 VP Platelet endothelial cell adhesion P16284 HR moleculePlatelet factor 4 P02776 VV Platelet-derived growth factor A P04085 JRP01127 Platelet-derived growth factor P04085 JQ Platelet-derived growthfactor P01127 HV subunit A (dimer) subunit B (dimer) Poly [ADP-ribose]polymerase 1 P09874 Kz, ** Pro-epidermal growth factor P01133 Lu, aU(cleaved) Pro-Interleukin-1 beta P01584- GH Pro-interleukin-16 Q14005Mv, bV Pro Prolactin P01236 IB Prostate-specific antigen P07288 CXProstatic acid phosphatase P15309 CV Protein NOV homolog P48745 UUProtein S100-A12 P80511 AX Protein S100-B P04271 XA Protransforminggrowth factor P01135 Mn, tX Renin P00797 Sr, sK alpha Resistin Q9HD89 WBSerum albumin P02768 St, zA Serum amyloid A protein P02735 HP Serumamyloid P-component P02743 Is, cZ Sex hormone-binding globulin P04278 DCSL cytokine P49771 LW Somatotropin P01241 BG Stromal cell-derived factor1 P48061 MS Stromelysin-1 P08254 CQ Stromelysin-l:MetalloproteinaseP08254 FI inhibitor 2 complex P16035 Stromelysin-2 P09238 UR TenascinP24821 TV Thrombomodulin P07204 Pj, hG Thrombopoietin P40225 Ne,d JThrombospondin-1 P07996 GW Thrombospondin-2 P35442 Uc, wP Thymic stromallymphopoietin Q969D9 NH Thyrotropin P01215 DK P01222 Thyroxine-bindingglobulin P05543 DD Tissue factor P13726 DE Tissue-type plasminogenactivator P00750 HL Transforming growth factor beta-1 P01137 Ql, qOTransforming growth factor beta-2 P61812 Qm, qP Transforming growthfactor beta-3 P10600 Qn, qQ Transmembrane glycoprotein Q14956 UNTransthyretin P02766 QH NMB Trefoil factor 3 Q07654 Ss, sMTubulointerstitial nephritis antigen Q9UJW2 GZ Tumor necrosis factorP01375 Oy, dH Tumor necrosis factor ligand P50591 NF superfamily member10 Tumor necrosis factor ligand O14788 YL Tumor necrosis factor ligandP48023 KR superfamily member 11 superfamily member 6 Tumor necrosisfactor receptor O14763 RZ Tumor necrosis factor receptor O00300 ONsuperfamily member 10B superfamily member 11B Tumor necrosis factorreceptor P19438 Kl, oQ Tumor necrosis factor receptor P20333 DGsuperfamily member 1A superfamily member 1B Tumor necrosis factorreceptor P25942 AP Tumor necrosis factor receptor P25445 KQ superfamilymember 5 superfamily member 6 Tumor necrosis factor receptor P28908 KCUrokinase plasminogen activator Q03405 UX superfamily member 8 surfacereceptor Urokinase-type plasminogen P00749 VA Vascular cell adhesionprotein 1 P19320 DL activator Vascular endothelial growth factor P15692Om, dM Vascular endothelial growth factor O43915 Ud, wQ A D Vascularendothelial growth factor P17948 KN Vascular endothelial growth factorP35968 KO receptor 1 receptor 2 Vascular endothelial growth factorP35916 KP Versican core protein P13611 HA receptor 3 Vitamin D-bindingprotein P02774 PS Vitamin K-dependent protein C P04070 GB von WillebrandFactor P04275 Iv, dN WAP four-disulfide core domain Q14508 Zq, zIprotein 2

The results of each analysis presented in the following examples arepresented in FIGS. 1-43.

Each figure contains an initial table presenting the univariatestatistics for the analytes that appear in the biomarker panels thatfollows in the same figure. The data set was segregated into two groups,non-diseased (NonDis) and diseased (Dis) patients as indicated in thefigure heading. The analyte code and units of measurement are shown,followed by the median, average, standard deviation, maximum and minimumof the analyte values calculated for each group. Also shown are thenumber of samples that make up each group, and the number of patientsthat the samples were drawn from. Values below the detectable limit of aparticular assay are indicated with the value 1×10⁻⁹ (written as“1.0E-9”). The univariate AUC for each analyte is also shown. Standarderrors were calculated as described in Hanley, J. A., and McNeil, B. J.,“The meaning and use of the area under a receiver operatingcharacteristic (ROC) curve,” Radiology (1982) 143: 29-36; p values werecalculated with a two-tailed Z-test. In the column specifying the unitsof measurement, certain analytes are listed as ‘2.6 ng/ml’, ‘2.5 ng/ml’,and ‘2.3 mU/ml’. For these markers, the values in the tables should bescaled by 2.6, 2.5, and 2.3 respectively. By way of example, a value of1 in the ‘median’ column converts to a value of 2.6 ng/ml, 2.5 ng/ml, or2.3 mU/ml, respectively. Note that applying such a scaling factor to thedata does not impact the AUC of a marker or the AUC of a panel of whichit is a part.

Following the univariate statistics, each figure provides panel tableswhich list the biomarker panels having model p-value in the rangesspecified in the particular figures. When a panel is formed by combiningmultiple analytes from this set, the number of samples used was theintersection of the samples measured by all analytes on the particularpanel. Panels that have an intersection of less than 7 samples in eitherthe non-diseased or diseased groups were not considered in the analysis.For compactness panels are encoded as follows: aBCdEE represents the twopanels aBCdEe and aBCdeE, in this case representing biomarker “ab”measured in plasma, biomarker “cd” measured in urine, and biomarker eemeasured in urine for the first panel and ee measured in plasma for thesecond panel. Codes in ( ) or { } and separated by a space represent alist of analytes that join with the analyte(s) outside the brackets toform panels with a common analyte. For example, Ab{cD(eF gH) Km(Ef QR)}represents 5 panels which all have biomarker Ab in common. They areAbcDeF, AbcDgH, AbKmEf, AbKmQr, and AbKmqR.

Those results labeled ‘Unconstrained Panels’ refer to panels which werenot required to have each analyte univariate p-value ≦0.05. Thoseresults labeled ‘Constrained Panels’ require that each analyte use inthe panel to have a univariate p-value ≦0.05.

EXAMPLE 7 Use of Biomarker Panels

Patients from the intensive care unit (ICU) were classified by kidneystatus as non-injury (0), risk of injury (R), injury (I), and failure(F) according to the maximum RIFLE stage reached as determined by theRIFLE criteria. Two cohorts were defined as (Cohort 1—“Non-diseased”)patients that did not progress beyond a particular RIFLE stage, and(Cohort 2—“Diseased”) patients that reached a later RIFLE stage within10 days. Marker concentrations were measured in samples collected from asubject at 0, 24 hours, and 48 hours prior to reaching the “Diseased”stage.

Each biomarker was measured by standard immunoassay methods usingcommercially available assay reagents. In the case of those biomarkerswhich are membrane-associated, assays which recognize soluble forms wereused. As noted above, the membrane-associated biomarkers includeSwiss-Prot entries O14788, O14944, O75309, P00797, P05186, P08473,P13688, P15514, P22223, P27487, P35070, Q03405, Q14956, Q16790, Q99075,Q9Y5Y7Q15109, Q02763, P17213, P12830, P33151, P06731, P29965, P16070,Q9H2A7, P17813, Q9UNN8, P00533, P16422, P19235, P16581, P78423, O43656,P08581, P08069, P05362, P13598, P32942, P14778, P27930, P01589, P24394,P08887, P40189, P21583, P09603, P08571, Q8WXI7, P13591, Q92823, P78380,P16284, P01133, P15309, P01135, P16109, Q14242, P49771, P07204, P13726,P01375, P50591, P48023, O14763, P19438, P20333, P25942, P25445, P28908,P19320, P17948, and P35968.

The ability of a biomarker panel to distinguish “Diseased” from“Non-Diseased” was determined as described above in Example 6. Patientsin Cohort 2 were also separated according to the reason for adjudicationto the “Diseased” stage as being based on serum creatinine measurements(sCr), being based on urine output (UO), or being based on either serumcreatinine measurements or urine output. As an example, for thosepatients adjudicated to stage F on the basis of serum creatininemeasurements alone, the stage 0 cohort may have included patientsadjudicated to stage F on the basis of urine output; for those patientsadjudicated to stage F on the basis of urine output alone, the stage 0cohort may have included patients adjudicated to stage F on the basis ofserum creatinine measurements; and for those patients adjudicated tostage F on the basis of serum creatinine measurements or urine output,the stage 0 cohort contains only patients in stage 0 for both serumcreatinine measurements and urine output. Also, for those patientsadjudicated to stage F on the basis of serum creatinine measurements orurine output, the adjudication method which yielded the most severeRIFLE stage was used.

The following data are presented in the figures:

-   FIG. 1. No or R RIFLE stage versus RIFLE I or F. RIFLE stage    adjudicated by sCr and Urine Output. Diseased group sample is 24    hours prior to RIFLE I diagnosis.-   FIG. 2. No or R RIFLE stage versus RIFLE I or F. RIFLE stage    adjudicated by sCr. Diseased group sample is 24 hours prior to RIFLE    I diagnosis.-   FIG. 3. No or R RIFLE stage versus RIFLE I or F. RIFLE stage    adjudicated by Urine Output. Diseased group sample is 24 hours prior    to RIFLE I diagnosis.-   FIG. 4. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage    adjudicated by sCr and Urine Output. Diseased group sample is 24    hours prior to RIFLE R diagnosis.-   FIG. 5. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage    adjudicated by sCr. Diseased group sample is 24 hours prior to RIFLE    R diagnosis.-   FIG. 6. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage    adjudicated by Urine Output. Diseased group sample is 24 hours prior    to RIFLE R diagnosis.-   FIG. 7. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage    adjudicated by sCr and Urine Output. Diseased group sample is 24    hours prior to RIFLE I diagnosis.-   FIG. 8. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage    adjudicated by Urine Output. Diseased group sample is 24 hours prior    to RIFLE I diagnosis.-   FIG. 9. No or R RIFLE stage versus RIFLE I or F. RIFLE stage    adjudicated by sCr and Urine Output. Diseased group sample is at    RIFLE I diagnosis.-   FIG. 10. No or R RIFLE stage versus RIFLE I or F. RIFLE stage    adjudicated by sCr. Diseased group sample is at RIFLE I diagnosis.-   FIG. 11. No or R RIFLE stage versus RIFLE I or F. RIFLE stage    adjudicated by Urine Output. Diseased group sample is at RIFLE I    diagnosis.-   FIG. 12. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage    adjudicated by sCr and Urine Output. Diseased group sample is at    RIFLE R diagnosis.-   FIG. 13. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage    adjudicated by sCr. Diseased group sample is at RIFLE R diagnosis.-   FIG. 14. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage    adjudicated by Urine Output. Diseased group sample is at RIFLE R    diagnosis.-   FIG. 15. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage    adjudicated by sCr and Urine Output. Diseased group sample is at    RIFLE I diagnosis.-   FIG. 16. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage    adjudicated by sCr. Diseased group sample is at RIFLE I diagnosis.-   FIG. 17. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage    adjudicated by Urine Output. Diseased group sample is at RIFLE I    diagnosis.-   FIG. 18. No or R RIFLE stage versus RIFLE I or F. RIFLE stage    adjudicated by sCr and Urine Output. Diseased group sample is 48    hours prior to RIFLE I diagnosis.-   FIG. 19. No or R RIFLE stage versus RIFLE I or F. RIFLE stage    adjudicated by sCr. Diseased group sample is 48 hours prior to RIFLE    I diagnosis.-   FIG. 20. No or R RIFLE stage versus RIFLE I or F. RIFLE stage    adjudicated by Urine Output. Diseased group sample is 48 hours prior    to RIFLE I diagnosis.-   FIG. 21. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage    adjudicated by sCr and Urine Output. Diseased group sample is 48    hours prior to RIFLE R diagnosis.-   FIG. 22. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage    adjudicated by sCr. Diseased group sample is 48 hours prior to RIFLE    R diagnosis.-   FIG. 23. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage    adjudicated by Urine Output. Diseased group sample is 48 hours prior    to RIFLE R diagnosis.-   FIG. 24. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage    adjudicated by sCr and Urine Output. Diseased group sample is 48    hours prior to RIFLE I diagnosis.-   FIG. 25. Progression of RIFLE R to RIFLE I or F. RIFLE stage    adjudicated by sCr and Urine Output. Sample is at R diagnosis.-   FIG. 26. Progression of RIFLE R to RIFLE I or F. RIFLE stage    adjudicated by sCr. Sample is at R diagnosis.-   FIG. 27. Progression of RIFLE R to RIFLE I or F. RIFLE stage    adjudicated by Urine Output. Sample is at R diagnosis.-   FIG. 28. No or R RIFLE stage versus RIFLE I or F. RIFLE stage    adjudicated by sCr and Urine Output, within 48 hrs of enrollment.    Sample is at enrollment.-   FIG. 29. No or R RIFLE stage versus RIFLE I or F. RIFLE stage    adjudicated by sCr, within 48 hrs of enrollment. Sample is at    enrollment.-   FIG. 30. No or R RIFLE stage versus RIFLE I or F. RIFLE stage    adjudicated by Urine Output, within 48 hrs of enrollment. Sample is    at enrollment.-   FIG. 31. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage    adjudicated by sCr and Urine Output, within 48 hrs of enrollment.    Sample is at enrollment.-   FIG. 32. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage    adjudicated by sCr, within 48 hrs of enrollment. Sample is at    enrollment.-   FIG. 33. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage    adjudicated by Urine Output, within 48 hrs of enrollment. Sample is    at enrollment.-   FIG. 34. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage    adjudicated by sCr and Urine Output, within 48 hrs of enrollment.    Sample is at enrollment.-   FIG. 35. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage    adjudicated by Urine Output, within 48 hrs of enrollment. Sample is    at enrollment.-   FIG. 36. No or R RIFLE stage versus RIFLE I or F. RIFLE stage    adjudicated by sCr and Urine Output, within 24 hrs of enrollment.    Sample is at enrollment.-   FIG. 37. No or R RIFLE stage versus RIFLE I or F. RIFLE stage    adjudicated by sCr, within 24 hrs of enrollment. Sample is at    enrollment.-   FIG. 38. No or R RIFLE stage versus RIFLE I or F. RIFLE stage    adjudicated by Urine Output, within 24 hrs of enrollment. Sample is    at enrollment.-   FIG. 39. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage    adjudicated by sCr and Urine Output, within 24 hrs of enrollment.    Sample is at enrollment.-   FIG. 40. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage    adjudicated by sCr, within 24 hrs of enrollment. Sample is at    enrollment.-   FIG. 41. No RIFLE stage versus RIFLE R, I, or F. RIFLE stage    adjudicated by Urine Output, within 24 hrs of enrollment. Sample is    at enrollment.-   FIG. 42. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage    adjudicated by sCr and Urine Output, within 24 hrs of enrollment.    Sample is at enrollment.-   FIG. 43. No, R, or I RIFLE stage versus RIFLE F. RIFLE stage    adjudicated by Urine Output, within 24 hrs of enrollment. Sample is    at enrollment.

EXAMPLE 8 Use of Biomarker Panels

The foregoing examples rely on logistic regression for identificationand use of biomarker panels. As noted, this is but one type of analysiswhich may be used to generate such panels of markers. Any classificationmethod can be used, including, but not limited to, Bayesian classifiers,discriminant analysis, decision trees, neural networks, support-vectormachines, nonparametric kernel density estimation methods, nearestneighbor rules, sums, differences, products and ratios of markerconcentrations.

As an example, the following exemplary biomarker panels of 2, 3, 4 and 5markers were generated using products (indicated by “*” in the table)and ratios (indicated by “I” in the table) of marker concentrations. Theproduct of the maker concentrations was used unless the panel consistedof markers that increased and markers that decreased with kidney injuryas determined by the univariate performance for the measured patientpopulation. In this case, a ratio was formed in which markers thatincreased were divided by markers that decreased.

In this example, Cohort 1 consisted of patients that did not progressbeyond RIFLE stage R and Cohort 2 consisted of patients that reachedstage I or F within 10 days. Panel values were determined for samplesfrom patients in Cohort 1 and samples drawn 24 (+/−12) hours prior tostage I (or F if no sample at stage I) in Cohort 2. Patients wereadjudicated to RIFLE stage R, I, or F based on either serum creatinineor urine output, whichever method yielded the most severe RIFLE stage. Areceiver operating characteristic (ROC) curve was generated for eachpanel and the area under each ROC curve (AUC) was determined. Standarderrors were calculated as described in Hanley, J. A., and McNeil, B. J.,The meaning and use of the area under a receiver operatingcharacteristic (ROC) curve. Radiology (1982) 143: 29-36; p values werecalculated with a two-tailed Z-test.

Examples of 2-, 3-, 4- and 5-marker panels are shown in the followingtable. The results for the 4 and 5 marker panels demonstrate that largerpanels can be formed with the kidney injury markers of Table 2, and thatthese panels can have improved p-values relative to 2 and 3-markerpanels.

TABLE 4 2-Marker Panels (AUC p-value < 1e-3) No/(dR) Nw*iO Fp/(Jj)No/(Ch) Lx*Nw Et*oZ Lx/(cM) No*aA No*iO Bc/(Jj) No/(Ck) No*Nw Lx*oZNw*cN Et*aA No/(iQ) MUD Gx*No Ok/(Jj) Nw*oZ No*cN No*eF Nw/(iQ) Kq/(Jj)nN/(mX) Ar*oF No*oZ Lx/(cW) Lx*eF Lj/(iT) No/(kR) Im*Lx Et/(oI) Nw*pANo/(cW) Nw*eF Is/(Bk) Nw/(kR) Kq*Lx No/(oI) Lj*pA Nw*cV Ar*eF Nw*iVLh/(Jj) Ky*Lx Lx/(oI) Mz*pA No*cV Ar/(eC) Nw/(iL) Lj/(Jj) Et*Lx Nw/(oI)No*pA Lx*cV Nw/(eC) Lx/(iL) Et*Lj Lx/(Kj) Nw/(oH) Ar*pA No*dC No/(eC)No/(iL) Mz/(Jj) Lx/(Jj) No/(oH) Ar*oY No/(dD) Mz/(eC) Lj/(iL) Lx*MzLx/(Ct) Lx/(oH) Mz*oY Lx/(dD) Et/(Bk) Ar/(iL) Lx/(Ng) Lx/(Ch) Lj/(oH)Et*oY Nw*gL Mz*iA No/(iH) Nr/(Jj) Lx/(Gp) Ar/(oH) Nw*oY No*gL Lj*iANw/(iH) No/(Jj) Bc*Lx Oh/(Jj) No*oY Mz/(gP) No*iA No*iK Jy*No Lx/(Aj)Nw*oL Et*Fp Et/(gP) No*hB Et*iK Im*No Fp*Lx No/(pC) bA*fR Ji/(gP) Nw*hBNw*iK Et*No Lx/(Az) Lx*pF cT*fR Nw/(gP) Mz*hB Et*Im No/(Kj) Lx/(Bk)Oa*pF cV*fR Lx/(gP) Lj*hB No/(iI) Mz*No Et*Mi Et*pF No*aJ No/(gP) Ar*hBNw/(iI) Lj*No No/(hH) Lj*pF Lx*aZ Ar/(gP) Et*hF Ar/(iI) Mi*No No*OaMz*pF Lx*bA Nw*hF Et*iZ Lw*No No/(nW) No*pF Lj*bA No*hF Nw*iX Lx*NoLx/(nW) Nw*pF By*bA Nw*hG Et*jA No/(Bk) Nw/(nW) Ar*pF Bm*bA Nw/(iR)Nw*jA Aw*No Et/(nW) Ar*pF Ar*bA No/(iR) Is/(Jj) Ay*No Ar/(nW) Ar*oZBb*bA Ar/(iR) Ij/(Jj) Fb*No Fp*Nw By*oZ Ba/(Aj) Ar*iP Im/(Jj) Fp*NoEt*Nw Nr*oZ Lj*bZ No*iP Ji/(Jj) No/(Aj) Nw/(Jj) Mz*oZ No*bZ Nw*iPEt/(Jj) By*No Im*Nw Lj*oZ No/(cK) 3-Marker Panels (AUC p-value < 1e-7)Et*Im/(Jj) No/(iI)/(Bk) Bc/(Jj)/(iR) No*Nw/(eC) Et*Lj/(iL) Et*No/(Bk)Et*No/(eC) No/(Bk)/(iJ) Ar*Nw/(iL) Et*oZ/(Ck) Lu*Im/(Jj) Ar*Nw*oZMz*Nw*pF Jy*Nw*iK No*oZ/(Jj) Et*Nw/(Bk) No/(gP)/(Kj) Is/(Bk)/(oH)No/(dR)/(Ch) oZ*Nw*pA No*eF/(Bk) No/(eC)/(cW) No/(nW)/(Ck) Lj/(Jj)/(iR)Fp*Nw*oZ Im*Nw/(Jj) Nw/(gP)/(Jj) No/(oH)/(Ch) Lj*Nw*pF Ar*Nw/(gP)No*cV/(Bk) Nw/(gP)/(Ck) oZ*No*pF No/(iR)/(eC) iK*No/(iL) Et*Nw/(gP)No/(oH)/(Ck) Nw*iK/(gP) Nw*iK/(Ck) Et*No/(iR) Et*No/(Jj) Mz*pF/(Jj)Mz*pF/(Bk) Nw*iK/(Jj) Ar*pA/(Ch) Et*Is/(Bk) Nw/(nW)/(Ck) Bc/(Jj)/(nW)Ar*Nw/(oH) Bc/(Jj)/(iQ) Lj*bZ/(Jj) No/(gP)/(Ck) No/(nW)/(Bk) Lu*Mz*pFNw/(iQ)/(Ck) No/(gP)/(Bk) Lj*oZ/(Jj) Lj/(oH)/(Jj) No*gL/(Bk) No*oZ/(Bk)Et*Im/(Bk) Et*Ar/(eC) Et/(gP)/(Jj) No*pF/(Bk) Ar*Et*oZ Im*No/(Jj)Et*No/(nW) No/(eC)/(Ch) Lj/(iL)/(Jj) No/(nW)/(gP) Et*No/(gP) oZ*Bc/(Jj)Jy*Nw/(gP) Et*iK/(Jj) Ar*oZ/(Jj) Et*Fp/(Jj) eF*Lj/(Jj) dC*Nw/(gP)No/(nW)/(Jj) No/(Bk)/(iS) No/(cW)/(Bk) No*pF/(Ch) No/(pC)/(eC)Lj/(iT)/(Jj) Ar*Et/(nW) No/(gP)/(Jj) No/(gP)/(cK) No/(iL)/(Bk)No/(gP)/(Ch) No/(nW)/(Ch) Pj*Im/(Jj) No*eF/(Jj) Et*Nw*hF Nw/(iR)/(eC)Et/(nW)/(Ck) Im*Ok/(Jj) aA*Nw/(eC) No/(iQ)/(Bk) Nw*pF/(Ch) No/(iI)/(Ch)No/(iR)/(Bk) Nw/(gP)/(aW) No/(eC)/(Jj) Et*Lj*oZ No/(gP)/(eC) No*Nw/(Bk)No*iK/(Jj) Lj*Nw*oZ Lu*No/(nW) Nw*pA/(Ch) Nw*oZ/(Ck) Lj/(Jj)/(gP)iK*Nw/(iL) Et*Lj*pF Ar*Nw/(iR) Et*Nw*iK Et*Nw*iO Im/(Jj)/(gP)Lj/(Jj)/(nW) Ar*Nw/(nW) Ij*Et/(Bk) aJ*Nw/(gP) No*oZ/(nW) Mz*pF/(Ch)No*pA/(Bk) No/(oH)/(Bk) No/(nW)/(eC) Ar*Nw/(eC) Nw/(oH)/(Ck)No/(iR)/(Ck) No*eF/(Ch) No*hB/(Bk) Mz*No/(gP) No*Nw/(nW) Ar/(oH)/(Jj)No*Nw/(gP) Lx/(gP)/(Ch) Mz*No*pF No*oZ/(Ck) Nw/(gP)/(eC) Bc/(Jj)/(oH)Nw/(gP)/(dD) Lw*Nw/(gP) No/(iR)/(Jj) iZ*Nw/(eC) Fy*Et/(Bk) fS*Lx/(Ch)Et*No*iK Et*Nw*pF 4-Marker Panels (AUC p-value < 1e-8) Nw*Im/Jj/KgOa*Et*Im/Jj Ar*Et*Im/Jj Mb*Nw*Im/Jj Et*Nw*Ji*Fp Nw*Im/Jj/Kj Fb*Nw*Im/JjPj*Et*Im/Jj Nt*Et*Im/Jj Fp*Nw*Im/Jj Ap*Nw*Im/Jj Hb*Et*Im/Jj Ke*Et*Im/JjIs*Et*Im/Jj Ji*Et*Im/Jj Bc*Nw*Im/Jj Bb*Et*Im/Jj Jy*Et*Im/Jj Nw*Ji*Fp/HwMb*Ji*Im/Jj Kp*Et*Im/Jj Cu*Et*Im/Jj Fb*Et*Im/Jj Mb*Et*Im/Jj Ji*Im/Jj/McJy*Nw*Im/Jj Bc*Et*Im/Jj Aw*Et*Im/Jj Et*Im/Jj/Mc Fp*Et*Im/Jj Cp*Nw*Im/JjEt*Im/Jj/Kg Nt*Nw*Im/Jj Ok*Et*Im/Jj Et*Ji*Fp/Jj Fy*Et*Im/Jj Ar*Ji*Im/JjNw*Im/Jj/Me Et*Nw*Im/Jj Fp*Ji*Im/Jj Pj*Nw*Im/Jj Aw*Nw*Im/Jj Is*Nw*Im/JjNw*Ji*Fp/Mc Et*Ji*Fp/Lu Kq*Nw*Im/Jj Et*Im/Jj/Kj Qe*Nw*Im/Jj Ji*Im/Jj/LuEt*Ji*Fp/Mc Ke*Nw*Im/Jj Aw*Ji*Im/Jj Nw*Im/Jj/Mc Ji*Nw*Im/Jj Nw*Ji*Fp/JjAr*Nw*Im/Jj Kq*Et*Im/Jj Ok*Nw*Im/Jj Ok*Ji*Im/Jj Nw*Ji*Fp/Lu Kk*Et*Im/JjCp*Et*Im/Jj Et*Aw*Qe/Jj Pj*Is/Jj/Kg Et*Ke*Isaj Et*Is*Ji/Jj Et*Ji*Nw/LuEt*Aw*Is/Jj Nt*Is*Mt/Ct Ny*Nm*Mt/Ct Et*Is*Fp/Jj Et*Jp*Fp/Jj Et*Is/Kj/JjNm*Pf*Mt/Ct Nm*Mt/Ct/Jj Et*Ji*Qe/Jj Et*Qe*Fp/Jj Im*Nm*Mt/Ct Qe*Cp*Et/JjNw*Et*Is/Kg Mb*Is*Ji/Jj Et*Fp/Mc/Jj Mm*Is/Jj/Kg Jy*Et*Qa/Jj Ji*Is/Jj/KgIs*Ji*Fp/Jj Im*Ok*Fp/Jj Ar*Et*Is/Jj Is*Nm*Mt/Ct Nm*Mt/Ct/Ch Et*Nw*Fp/JjJi*Jp*Fp/Jj Qa*Et*Aw/Jj Nt*Pf*Mt/Ct Is*Ok/Jj/Kg Et*Ok*Fp/Jj Ji*Qe*Fp/JjJy*Et*Qe/Jj Nb*Pf*Mt/Ct Mz*Nm*Mt/Ct Et*On*Fp/Jj Ji*On*Fp/Jj Is*Im*Mt/CtIs*Pf*Mt/Ct Ke*Is/Jj/Kg Ji*Ok*Fp/Jj Et*Nw*Fp/Mc Nb*Is*Mt/Ct Ji*Et*Aw/JjKe*Et*Fp/Jj Et*Nw*Fp/Lu Ok*Fp/Jj/Mc Et*Is*Mt/Ct Im*Pf*Mt/Ct Im*Is/Jj/KgNw*Ok*Fp/Jj Et*Is/Jj/Kg Et*Im*Ji/Aj Nt*Im*Mt/Ct Is*Nw/Jj/Kg 5-MarkerPanels (AUC p-value < 1e-9) Mz*Nw*Im/Jj/Kg Lh*Et*Im/Jj/Kg Nt*Fp*Nw*Im/JjJp*Fp*Nw*Im/Jj Kq*Nw*Im/Jj/Kg Ip*Nw*Im/Jj/Kg Mb*Ok*Nw*Ji*FpMz*Fp*Nw*Im/Jj Qa*Nw*Im/Jj/Kg Dg*Ji*Im/Jj/Kj Et*Is*Nw*Ji*FpNt*Nw*Ji*Fp/Jj Ma*Nw*Ji*Fp/Aj Et*Cp*Ji*Im/Jj On*Nw*Ji*Fp/HwJt*Nw*Ji*Fp/Jj Ok*Nw*Im/Jj/Kg Nm*Et*Im/Jj/Aj Et*Mi*Nw*Ji*FpMi*Nw*Ji*Fp/Jj Ji*Nw*Im/Jj/Kg Et*Cp*Nw*Im/Jj Qe*Fp*Nw*Im/JjPz*Nw*Ji*Fp/Jj Ke*Aw*Nw*Im/Jj Et*Jy*Ji*Im/Jj Fp*Nw*Im/Jj/HwJp*Nw*Ji*Fp/Lu Ip*Nw*Im/Jj/Kj Ad*Nw*Im/Jj/Kj Is*Fp*Nw*Im/JjMb*Nw*Ji*Fp/Lu Bc*Nw*Im/Jj/Kj Et*Ar*Nw*Im/Jj Im*Nw*Ji*Fp/McMz*Et*Ji*Fp/Hw Ef*Nw*Im/Jj/Aj Kl*Ji*Im/Jj/Aj Lw*Nw*Ji*Fp/JjIs*Et*Ji*Fp/Hw Ok*Nw*Im/Jj/Kj Jo*Et*Im/Jj/Kg Jo*Nw*Ji*Fp/JjOk*Et*Ji*Fp/Hw Ap*Et*Ji*Fp/Jj Ad*Nw*Im/Jj/Aj Js*Nw*Ji*Fp/JjEt*Is*Ji*Im/Jj Qe*Nw*Im/Jj/Kj Kq*Et*Im/Jj/Kj Jp*Nw*Ji*Fp/McIs*Ji*Im/Jj/Hw Nt*Aw*Nw*Im/Jj Dl*Et*Im/Jj/Aj Qe*Nw*Ji*Fp/LuIs*Et*Ji*Fp/Jj Fb*Aw*Nw*Im/Jj Bc*Et*Im/Jj/Kj Lw*Nw*Ji*Fp/LuEt*Fp*Nw*Im/Jj Pj*Nw*Im/Jj/Kj Ok*Et*Im/Jj/Kg Mb*Nw*Ji*Fp/McEt*Ji*Nw*Im/Jj Ip*Nw*Im/Jj/Aj Qe*Et*Im/Jj/Kj Et*Nt*Nw*Im/JjEt*Nw*Im/Jj/Lu Qe*Nw*Im/Jj/Kg Ke*Et*Im/Jj/Ob Et*Ok*Nw*Ji*FpEt*Nw*Im/Jj/Mc Jg*Nw*Im/Jj/Aj Kq*Aw*Et*Im/Jj Jp*Nw*Ji*Fp/HwEt*Mb*Nw*Im/Jj Kq*Aw*Nw*Im/Jj Ke*Aw*Et*Im/Jj Et*Jp*Nw*Ji*FpJi*Et*Im/Jj/Lu Et*Nw*Im/Jj/Aj Mm*Et*Im/Jj/Kj Nw*Ji*Fp/Nh/JjJi*Fp*Et*Im/Jj Ad*Et*Ji*Fp/Aj Mm*Et*Im/Jj/Kg Ma*Nw*Ji*Fp/JjJi*Et*Im/Jj/Mc Ad*Et*Ji*Fp/Jj Ke*Et*Im/Jj/Kg Lh*Nw*Ji*Fp/JjJi*Mb*Et*Im/Jj Jg*Et*Ji*Fp/Aj Kq*Et*Im/Jj/Aj Mz*Nt*Nw*Ji*FpOk*Fp*Et*Im/Jj Et*Kk*Ji*Im/Jj Ad*Ji*Im/Jj/Aj Ik*Ji*Nw*Im/JjFp*Et*Im/Jj/Mc Pj*Ji*Im/Jj/Kg Jp*Aw*Et*Im/Jj Nt*Et*Ji*Fp/LuEt*Nt*Ji*Im/Jj Is*Ji*Im/Jj/Kg Ik*Ji*Im/Jj/Aj Nt*Nw*Et*Ji*FpFp*Ji*Im/Jj/Hw Ok*Aw*Nw*Im/Jj Kq*Et*Im/Jj/Ch Mi*Nw*Ji*Fp/HwEt*Ok*Ji*Im/Jj Pf*Nw*Im/Jj/Ct Dg*Ji*Im/Jj/Aj Nw*Ji*Fp/Me/HwEt*Jp*Ji*Im/Jj Mb*Aw*Nw*Im/Jj Ef*Et*Im/Jj/Aj Nw*Ji*Fp/Mc/HwEt*Qe*Ji*Im/Jj Dg*Et*Ji*Fp/Aj Ar*Et*Im/Jj/Mc Qe*Nw*Ji*Fp/HwEt*Mi*Ji*Im/Jj Aw*Nw*Im/Jj/Ob Nt*Aw*Et*Im/Jj Qa*Nw*Ji*Fp/HwIm*Et*Ji*Fp/Lu Jp*Aw*Nw*Im/Jj Et*Nw*Im/Jj/Kj Mb*Fp*Nw*Im/JjJg*Et*Ji*Fp/Jj Et*Cu*Nw*Im/Jj Qa*Et*Im/Jj/Kg Nw*Ji*Fp/Me/JjJi*Fp*Nw*Im/Jj Et*Oa*Nw*Im/Jj Pj*Et*Im/Jj/Kg Om*Nw*Ji*Fp/JjJi*Nw*Im/Jj/Lu Bb*Et*Ji*Fp/Jj Pj*Et*Im/Jj/Mc Mi*Nw*Ji*Fp/LuJi*Mb*Nw*Im/Jj Pj*Nw*Im/Jj/Kg Ke*Et*Im/Jj/Mc Ok*Nw*Ji*Fp/McJi*Nw*Im/Jj/Mc Mt*Nw*Im/Jj/Ct Lw*Fb*Et*Im/Jj Qe*Et*Ji*Fp/HwFp*Ji*Im/Jj/Mc Nw*Im/Jj/Ob/Mc Pj*Et*Im/Jj/Kj Qa*Et*Ji*Fp/HwOk*Fp*Ji*Im/Jj Et*Kk*Nw*Im/Jj Ik*Nw*Im/Jj/Aj 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While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention. The examplesprovided herein are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Modifications therein and other uses will occur to thoseskilled in the art. These modifications are encompassed within thespirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

We claim:
 1. A method for evaluating biomarker levels in a plasma orurine sample from a subject, comprising: obtaining a plasma or urinesample from a subject that suffers from current acute renal injury;performing a plurality of assays comprising a first assay configured todetect Beta-2-microglobulin and a second assay configured to detect WAPfour-disulfide core domain protein 2 by introducing the plasma or urinesample obtained from the subject into an assay instrument which (i) foreach analyte binding assay performed, contacts all or a portion of theplasma or urine sample with a binding reagent which specifically bindsfor detection the kidney injury marker which is assayed, and (ii)generates a first assay result indicative of binding ofBeta-2-microglobulin to its respective binding reagent and a secondassay result indicative of binding of WAP four-disulfide core domainprotein 2 to its respective binding reagent; combining the first andsecond assay results using a function that converts the first and secondassay results into a single composite result; and correlating the singlecomposite result to a likelihood that the renal function of the subjectwill improve within 72 hours of the time at which the plasma or urinesample is obtained from the subject by using the single composite resultto assign the subject to a predetermined subpopulation of individualshaving a current acute renal injury and that have a known predispositionto the nonoccurrence of acute renal failure within 72 hours.
 2. A methodaccording to claim 1, wherein said correlating step comprisescorrelating the single composite result to a likelihood that the renalfunction of the subject will improve within 48 hours of the time atwhich the plasma or urine sample is obtained.
 3. A method according toclaim 1, wherein said correlating step comprises correlating the singlecomposite result to a likelihood that the renal function of the subjectwill improve within 24 hours of the time at which the plasma or urinesample is obtained.
 4. A method according to claim 1, wherein thesubject is in RIFLE stage I or F.
 5. A method according to claim 2,wherein the subject is in RIFLE stage I or F.
 6. A method according toclaim 3, wherein the subject is in RIFLE stage I or F.